Publications

  • Urin V., Shemesh M. & Schreiber G. 2019. CRISPR/Cas9-based Knockout Strategy Elucidates Components Essential for Type 1 Interferon Signaling in Human HeLa Cells Journal of Molecular Biology. 2019 Aug , 431 (17):3324-3338.

    [Abstract]

    Type I interferons (IFNs) have a central role in innate and adaptive immunities, proliferation, and cancer surveillance. How IFN binding to its specific receptor, the IFN alpha and beta receptor (IFNAR), can drive such variety of processes is an open question. Here, to systematically and thoroughly investigate the molecular mechanism of IFN signaling, we used a CRISPR/Cas9-based approach in a human cell line (HeLa) to generate knockouts (KOs) of the genes participating in the type 1 IFN signaling cascade. We show that both IFNAR chains (IFNAR1 and IFNAR2) are absolutely required for any IFN-induced signaling. Deletion of either signal transducer and activator of transcription 1 (STAT1) or STAT2 had only a partial effect on IFN-induced antiviral activity or gene induction. However, the deletion of both genes completely abrogated any IFN-induced activity. So did a double STAT2 IFN regulatory factor 1 (IRF1) KO and, to a large extent, a STAT1 KO together with IRF9 knockdown. KO of any of the STATs had no effect on the phosphorylation of other STATs, indicating that they bound IFNAR independently. STAT3 and STAT6 phosphorylations were fully induced by type 1 IFN in the STAT1 STAT2 KO, but did not promote gene induction. Moreover, STAT3 KO did not affect type 1 IFN-induced gene or protein expression. Type 1 IFN also did not activate p38, AKT, or ERK kinase. We conclude that type 1 IFN-induced activities in HeLa cells are mediated by STAT1/STAT2/IRF9, STAT1/STAT1, or STAT2/IRF9 complexes and do not require alternative pathways. (C) 2019 Elsevier Ltd. All rights reserved.
  • Schlaepfer E., Fahrny A., Gruenbach M., Kuster S. P., Simon V., Schreiber G. & Speck R. F. 2019. Dose-Dependent Differences in HIV Inhibition by Different Interferon Alpha Subtypes While Having Overall Similar Biologic Effects mSphere. 2019 Feb , 4 (1).

    [Abstract]

    Type I interferons (IFNs) are key players in the antiviral immune response. Interferon alpha (IFN-alpha) belongs to this class of IFNs and comprises 12 subtypes that differ from each other in their binding affinities for a common receptor and, thus, in their signaling potencies. Recent data suggest that IFN-alpha 6 and -alpha 14 are the most potent IFN-alpha subtypes in restricting HIV replication when applied exogenously. However, in the context of antiviral therapy, IFNs are administered at high doses, which may compensate for differences in potency seen between IFN-alpha subtypes. In this study, we reexamined whether IFN-alpha subtypes induce different biological activities, with a focus on how IFN-alpha treatment dose affects cellular responses to HIV in primary CD4+ T cells, peripheral blood mononuclear cells (PBMCs), and macrophages. We found that the subtypes' antiviral activities were dose dependent, with >90% inhibition of HIV replication at a high dose of all IFN-alpha s except the weak IFN-alpha/beta receptor (IFNAR) binder, IFN-alpha 1. The quality of the responses engendered by IFN-alpha 1, -alpha 2, -alpha 6, and -alpha 14 was highly comparable, with essentially the same set of genes induced by all four subtypes. Hierarchal cluster analysis revealed that the individual donors were stronger determinants for the IFN-stimulated-gene (ISG) responses than the specific IFN-alpha subtype used for stimulation. Notably, IFN-alpha 2-derived mutants with substantially reduced IFNAR2 binding still inhibited HIV replication efficiently, whereas mutants with increased IFNAR1 binding potentiated antiviral activity. Overall, our results support the idea that IFN-alpha subtypes do not induce different biological responses, given that each subtype is exogenously applied at bioequivalent doses.IMPORTANCE Elucidating the functional role of the IFN-alpha subtypes is of particular importance for the development of efficacious therapies using exogenous IFN-alpha. Specifically, this will help define whether IFN therapy should be based on the use of pathogen-dependent IFN subtypes or, rather, IFN mutants with optimized IFNAR binding properties.
  • Cohen-Khait R. & Schreiber G. 2018. Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP Biochemistry. 2018 Aug , 57 (31):4644-4650.

    [Abstract]

    Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-beta-lactamase library against its natural nanomolar affinity binder beta-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.
  • Nganou-Makamdop K., Billingsley J. M., Yaffe Z., O'Connor G., Tharp G. K., Ransier A., Laboune F., Matus-Nicodemos R., Lerner A., Gharu L., Robertson J. M., Ford M. L., Schlapschy M., Kuhn N., Lensch A., Lifson J., Nason M., Skerra A., Schreiber G., Bosinger S. E. & Douek D. C. 2018. Type I IFN signaling blockade by a PASylated antagonist during chronic SIV infection suppresses specific inflammatory pathways but does not alter T cell activation or virus replication PLoS Pathogens. 2018 Aug , 14 (8).

    [Abstract]

    Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naive macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1 beta, IL-lra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-1-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.
  • Baeuerle F., Zotter A. & Schreiber G. 2017. Direct determination of enzyme kinetic parameters from single reactions using a new progress curve analysis tool PROTEIN ENGINEERING DESIGN & SELECTION. 2017 Mar , 30 (3):151-158.
  • Schreiber G. 2017. The molecular basis for differential type I interferon signaling Journal of Biological Chemistry. 2017 May , 292 (18):7285-7294.
  • Li H., Sharma N., General I. J., Schreiber G. & Bahar I. 2017. Dynamic Modulation of Binding Affinity as a Mechanism for Regulating Interferon Signaling Journal of Molecular Biology. 2017 Aug , 429 (16):2571-2589.

    [Abstract]

    How structural dynamics affects cytokine signaling is under debate. Here, we investigated the dynamics of the type I interferon (IFN) receptor, IFNAR1, and its effect on signaling upon binding IFN and IFNAR2 using a combination of structure-based mechanistic studies, in situ binding, and gene induction assays. Our study reveals that IFNAR1 flexibility modulates ligand-binding affinity, which, in turn, regulates biological signaling. We identified the hinge sites and key interactions implicated in IFNAR1 inter-subdomain (SD1-SD4) movements. We showed that the predicted cooperative movements are essential to accommodate intermolecular interactions. Engineered disulfide bridges, computationally predicted to interfere with IFNAR1 dynamics, were experimentally confirmed. Notably, introducing disulfide bonds between subdomains SD2 and SD3 modulated IFN binding and activity in accordance with the relative attenuation of cooperative movements with varying distance from the hinge center, whereas locking the SD3 SD4 interface flexibility in favor of an extended conformer increased activity. (C) 2017 Elsevier Ltd. All rights reserved.
  • Cohen-Khait R., Dym O., Hamer-Rogotner S. & Schreiber G. 2017. Promiscuous Protein Binding as a Function of Protein Stability Structure (London, England : 1993). 2017 Dec , 25 (12):1867-+.

    [Abstract]

    Proteins have evolved to balance efficient binding of desired partners with rejection of unwanted interactions. To investigate the evolution of protein-protein interactions, we selected a random library of pre-stabilized TEM1 beta-lactamase against wild-type TEM1 using yeast surface display. Three mutations were sufficient to achieve micromolar affinity binding between the two. The X-ray structure emphasized that the main contribution of the selected mutations was to modify the protein fold, specifically removing the N'-terminal helix, which consequently allowed protein coupling via a beta-sheet-mediated interaction resembling amyloid interaction mode. The only selected mutation located at the interaction interface (E58V) is reminiscent of the single mutation commonly causing sickle-cell anemia. Interestingly, the evolved mutations cannot be inserted into the wild-type protein due to reduced thermal stability of the resulting mutant protein. These results reveal a simple mechanism by which undesirable binding is purged by loss of thermal stability.
  • Zotter A., Baeuerle F., Dey D., Kiss V. & Schreiber G. 2017. Quantifying enzyme activity in living cells Journal of Biological Chemistry. 2017 Sept , 292 (38):15838-15848.

    [Abstract]

    For over a century, enzymatic activity has been studied in vitro, assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-beta-lactamase inside living cells and compared the values to those obtained in vitro. We found the apparent in vivo catalytic efficiency, k(cat)/K-m, to be lower than in vitro, with significant cell-tocell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases, and Km increases with increasing enzyme concentration. To rationalize these findings, we measured enzyme and substrate diffusion rates in the cell and found the latter to be slower than expected. Simulations showed that for attenuated diffusion the substrate flux becomes rate-limiting, explaining why reaction rates in vivo can be independent on enzyme concentrations. The octanol/water partition of the substrate is 4.5, which is in the range of Food and Drug Administration-approved drugs. This suggests substrate-limited reaction rates to be common. These findings indicate that in vitro data cannot be simply extrapolated to the crowded in vivo environment.
  • Sharma N., Longjam G. & Schreiber G. 2016. Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain Journal of Biological Chemistry. 2016 Feb , 291 (7):3371-3384.

    [Abstract]

    Type I interferons serve as the first line of defense against pathogen invasion. Binding of IFNs to its receptors, IFNAR1 and IFNAR2, is leading to activation of the IFN response. To determine whether structural perturbations observed during binding are propagated to the cytoplasmic domain, multiple mutations were introduced into the transmembrane helix and its surroundings. Insertion of one to five alanine residues near either the N or C terminus of the transmembrane domain (TMD) likely promotes a rotation of 100 degrees and a translation of 1.5 per added residue. Surprisingly, the added alanines had little effect on the binding affinity of IFN to the cell surface receptors, STAT phosphorylation, or gene induction. Similarly, substitution of the juxtamembrane residues of the TMD with alanines, or replacement of the TMD of IFNAR1 with that of IFNAR2, did not affect IFN binding or activity. Finally, only the addition of 10 serine residues (but not 2 or 4) between the extracellular domain of IFNAR1 and the TMD had some effect on signaling. Bioinformatic analysis shows a correlation between high sequence conservation of TMDs of cytokine receptors and the ability to transmit structural signals. Sequence conservation near the TMD of IFNAR1 is low, suggesting limited functional importance for this region. Our results suggest that IFN binding to the extracellular domains of IFNAR1 and IFNAR2 promotes proximity between the intracellular domains and that differential signaling is a function of duration of activation and affinity of binding rather than specific conformational changes transmitted from the outside to the inside of the cell.
  • Cohen-Khait R. & Schreiber G. 2016. Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders Proceedings of the National Academy of Sciences of the United States of America. 2016 Dec , 113 (52):14982-14987.

    [Abstract]

    Protein-protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 beta-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the libraryligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes.
  • Schreiber G. & Piehler J. 2015. The molecular basis for functional plasticity in type I interferon signaling Trends in Immunology. 2015 Mar , 36 (3):139-149.

    [Abstract]

    Type I interferons (IFNs) are best known for their role in innate immunity, but they are also involved in other functions including immunomodulation, restricting proliferation, cancer surveillance, and the regulation of the adaptive immune response. All these responses are mediated through the interaction with a single cell surface receptor, albeit at different ligand and receptor concentrations, ligand subtypes, and time of activation. Here we review the functional plasticity of IFN signaling from a quantitative perspective, showing how variations in different ingredients of the system lead to differential IFN responses and how cells tune the system to maximize efficiency while minimizing detrimental effects. We present a basic model wherein the integrated action of different feedback mechanisms can provide sufficient temporal control to differentially drive cellular decisions.
  • Urin V., Levin D., Sharma N., Harari D. & Schreiber G. 2015. Fine Tuning of a Type 1 Interferon Antagonist PLoS One. 2015 Jul , 10 (7).

    [Abstract]

    Type I interferons are multi-potent cytokines that serve as first line of defense against viruses and other pathogens, posses immunomudolatory functions and elicit a growth inhibitory response. In recent years it has been shown that interferons are also detrimental, for example in lupus, AIDS, tuberculosis and cognitive decline, highlighted the need to develop interferon antagonists. We have previously developed the antagonist IFN-1ant, with much reduced binding to the IFNAR1 receptor and enhanced binding to IFNAR2. Here, we further tune the IFN-1ant by producing three additional antagonists based on IFN-1ant but with altered activity profiles. We show that in all three cases the antiproliferative activity of interferons is blocked and the induction of gene transcription of immunomudolatory and antiproliferative associated genes are substantially decreased. Conversely, each of the new antagonists elicits a different degree of antiviral response, STAT phosphorylation and related gene induction. Two of the new antagonists promote decreased activity in relation to the original IFN-1ant, while one of them promotes increased activity. As we do not know the exact causes of the detrimental effects of IFNs, the four antagonists that were produced and analyzed provide the opportunity to investigate the extent of antagonistic and agonistic activity optimal for a given condition.
  • Harari D., Orr I., Rotkopf R., Baranzini S. & Schreiber G. 2015. A robust type I interferon gene signature from blood RNA defines quantitative but not qualitative differences between three major IFN beta drugs in the treatment of multiple sclerosis Human Molecular Genetics. 2015 Jun , 24 (11):3192-3205.

    [Abstract]

    We analysed gene expression microarray data from whole blood samples from 228 multiple sclerosis (MS) patients either untreated or treated with one of three alternative commonly used interferon beta (IFN beta) disease modifying drugs: Avonex(A (R)) (x1 weekly), Betaseron(A (R)) (every second day) or Rebif(A (R)) (x3 weekly). Patient injections were not timed to coordinate sample collections, thus providing a global transcriptomic profile for each population of patients studied. Three hundred and fifty one genes were significantly differentially expressed by at least one of the IFN beta drugs. Despite the different drug sources with distinct injection and dosage protocols, a striking similarity was found in the identity and functional classes of the differentially expressed genes induced. Using the 25 most-upregulated genes, we defined a robust IFN beta gene expression signature that quantifies the IFN activation state per blood sample collected irrespective of the type of IFN beta therapy. This 25-gene signature also defined basal IFN activation states among untreated MS patients, which differed among individuals but remained relatively constant per patient with time. The maximum drug-induced IFN-activation state was similar for all three drugs despite a 1.7-2.0-fold diminished average effect for Avonex. This and a more erratic effect of Avonex per patient across longitudinal measurements is likely a result of its reduced injection frequency. In summary, we have defined a robust blood-derived type I IFN gene signature from MS patients. This signature could potentially serve to generically quantify the systemic Type I IFN activation status for any other clinical manifestation, inclusive of other autoimmune diseases.
  • Schreiber G. & VanHook A. M. 2014. Science Signaling Podcast: 27 May 2014 Science Signaling. 2014 May , 7 (327).

    [Abstract]

    This Podcast features an interview with Gideon Schreiber, senior author of a Research Article that appears in the 27 May 2014 issue of Science Signaling, about an engineered variant of a type I interferon that elicits only antiviral responses. Interferons are major components of the innate immune response and serve as a link between the innate and adaptive immune responses. Type I interferons stimulate the expression of many genes that affect various cellular processes, including limiting the spread of viruses between cells, suppressing cell proliferation, and modulating the immune response. Levin et al. identified a variant of the type I interferon IFN-alpha 2 that elicits antiviral responses without suppressing cell proliferation or activating immunomodulatory genes.
  • Tiberti M., Invernizzi G., Lambrughi M., Inbar Y., Schreiber G. & Papaleo E. 2014. PyInteraph: A Framework for the Analysis of Interaction Networks in Structural Ensembles of Proteins Journal of Chemical Information and Modeling. 2014 May , 54 (5):1537-1551.

    [Abstract]

    In the last years, a growing interest has been gathering around the ability of Molecular Dynamics (MD) to provide insight into the paths of long-range structural communication in biomolecules. The knowledge of the mechanisms related to structural communication helps in the rationalization in atomistic details of the effects induced by mutations, ligand binding, and the intrinsic dynamics of proteins. We here present PyInteraph, a tool for the analysis of structural ensembles inspired by graph theory. PyInteraph is a software suite designed to analyze MD and structural ensembles with attention to binary interactions between residues, such as hydrogen bonds, salt bridges, and hydrophobic interactions. PyInteraph also allows the different classes of intra- and intermolecular interactions to be represented, combined or alone, in the form the resulting interaction graphs. The program also integrates of interaction graphs, along with performing network analysis on the network description with a knowledge-based force field to estimate the interaction energies between side chains in the protein. It can be used alone or together with the recently developed xPyder PyMOL plugin through an xPyder-compatible format. The software capabilities and associated protocols are here illustrated by biologically relevant cases of study. The program is available free of charge as Open Source software via the GPL v3 license at http://linumbtbs.unimibit/pyinteraph/.
  • Harari D., Kuhn N., Abramovich R., Sasson K., Zozulya A. L., Smith P., Schlapschy M., Aharoni R., Koester M., Eilam R., Skerra A. & Schreiber G. 2014. Enhanced in Vivo Efficacy of a Type I Interferon Superagonist with Extended Plasma Half-life in a Mouse Model of Multiple Sclerosis Journal of Biological Chemistry. 2014 Oct , 289 (42):29014-29029.

    [Abstract]

    IFN beta is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNS alpha 8) that is built on the backbone of a low affinity IFN alpha but modified to exhibit higher receptor affinity than even for IFN beta. Here, YNS alpha 8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNS alpha 8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG(35-55)-induced experimental autoimmune encephalomyelitis compared with IFN beta, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNS alpha 8-treated animals. Importantly, PAS-YNS alpha 8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.
  • Bardwell J. & Schreiber G. 2014. Editorial overview: Folding and binding Current Opinion in Structural Biology. 2014 Feb , 24 :VIII-X.
  • Harari D., Abramovich R., Zozulya A., Smith P., Pouly S., Koester M., Hauser H. & Schreiber G. 2014. Bridging the Species Divide: Transgenic Mice Humanized for Type-I Interferon Response PLoS One. 2014 Jan , 9 (1).

    [Abstract]

    We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These "HyBNAR" (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFN alpha 2A, IFN beta, and a human superagonist termed YNS alpha 8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.
  • Sandler N. G., Bosinger S. E., Estes J. D., Zhu R. T. R., Tharp G. K., Boritz E., Levin D., Wijeyesinghe S., Makamdop K. N., del Prete P. G. Q., Hill B. J., Timmer J. K., Reiss E., Yarden G., Darko S., Contijoch E., Todd J. P., Silvestri G., Nason M., Norgren R. B. J., Keele B. F., Rao S., Langer J. A., Lifson J. D., Schreiber G. & Douek D. C. 2014. Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression Nature. 2014 Jul , 511 (7511):601-+.

    [Abstract]

    Inflammation in HIV infection is predictive of non-AIDS morbidity and death(1), higher set point plasma virus load(2) and virus acquisition3; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection(4-10), also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression(6,11-19). Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (Sly) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-a2a administration initially upreg-ulated expression of antiviral genes and prevented systemic infection. However, continued IFN-a2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.
  • Harari D., Kallweit N., Abramovich R., Sasson K., Zozulya A., Smith P., Schlapschy M., Aharoni R., Koester M., Eliam R., Skerra A. & Schreiber G. 2014. Enhanced in vivo efficacy of a long-life type I Interferon superagonist in a mouse model of multiple sclerosis Journal of Neuroimmunology. 2014 Oct , 275 (2-Jan):219-219.

    [Abstract]

    IFN-Beta (IFNB) has been a major therapeutic option to treat multiple sclerosis (MS) for more than two decades. Newer drugs have more recently been accepted for patient use with clinical trial data often suggesting superior therapeutic efficacy but with the price of a less favorable safety profile. We have recently reported the generation of an interferon superagonist (dubbed YNSa8) that behaves like a potent form of IFNB as demonstrated in transgenic mice harboring humanized receptors to the Type-I Interferons. For this protein we have used a novel technology named “PASylation” to convert YNSa8 into a long-acting drug when injected into animals. PAS-YNSa8 demonstrated a 10-fold increased half-life in mice, but notably without any detectable loss in biological potency or bioavailability. Furthermore, in comparison to both human and mouse IFNB, this long-living superagonist conferred improved protection from MOG35–55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a mouse model commonly used to emulate human MS. The improved therapeutic effect of PAS-YNSa8 relative to IFNB took place despite the 4-fold decrease in frequency of injection and at an overall 16-fold lower dosage used. The phenotypic activity of this improved functioning IFN-variant is in line with that published by others regarding the role of IFN signaling in EAE — We detected a decrease in monocytes, possibly infiltrating macrophages into the CNS as a consequence to IFN therapy. Furthermore, a direct correlation between improvement in IFN-therapeutic response and the upregulation of the T-cell immune-tolerance factor PD-L1 and the chemokine receptor CXCR3 were detected in CD4-isolated spleen cells, implicating these factors as major player in transducing the therapeutic response to IFN therapy. The possible implications of our findings for treating MS will be discussed.
  • Levin D., Schneider W. M., Hoffmann H., Yarden G., Busetto A. G., Manor O., Sharma N., Rice C. M. & Schreiber G. 2014. Multifaceted Activities of Type I Interferon Are Revealed by a Receptor Antagonist Science Signaling. 2014 May , 7 (327).

    [Abstract]

    Type I interferons (IFNs), including various IFN-alpha isoforms and IFN-beta, are a family of homologous, multifunctional cytokines. IFNs activate different cellular responses by binding to a common receptor that consists of two subunits, IFNAR1 and IFNAR2. In addition to stimulating antiviral responses, they also inhibit cell proliferation and modulate other immune responses. We characterized various IFNs, including a mutant IFN-alpha 2 (IFN-1ant) that bound tightly to IFNAR2 but had markedly reduced binding to IFNAR1. Whereas IFN-1ant stimulated antiviral activity in a range of cell lines, it failed to elicit immuno-modulatory and antiproliferative activities. The antiviral activities of the various IFNs tested depended on a set of IFN-sensitive genes (the "robust" genes) that were controlled by canonical IFN response elements and responded at low concentrations of IFNs. Conversely, these elements were not found in the promoters of genes required for the antiproliferative responses of IFNs (the "tunable" genes). The extent of expression of tunable genes was cell type-specific and correlated with the magnitude of the antiproliferative effects of the various IFNs. Although IFN-1ant induced the expression of robust genes similarly in five different cell lines, its antiviral activity was virus-and cell type-specific. Our findings suggest that IFN-1ant may be a therapeutic candidate for the treatment of specific viral infections without inducing the immunomodulatory and antiproliferative functions of wild-type IFN.
  • Harari D., Kallweit N., Abramovich R., Sasson K., Zozulya A., Smith P., Schlapschy M., Aharoni R., Koester M., Eliam R., Skerra A. & Schreiber G. 2014. Enhanced in vivo efficacy using a novel long-life type I interferon variant in a mouse model of multiple sclerosis Cytokine. 2014 Nov , 70 (1):45-45.

    [Abstract]

    IFN is a common therapeutic option to treat multiple sclerosis (MS). It is unique amongst the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNS8) that is built on the backbone of a low affinity IFN, but modified to exhibit higher receptor affinity than IFN. Here, YNS8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain, comprising Proline, Alanine and Serine (PAS), in order to prolong its plasma half-life via “PASylation”. PAS-IFN8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors [1], notably without any detectable loss in biological potency or bioavailability. This long-life superagonist conferred significantly improved protection from MOG35–55 peptide-induced experimental autoimmune encephalomyelitis (EAE) compared to IFN, despite being injected with a 4-fold decrease in frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS and immunohistochemistry, showing a decrease of myeloid lineage cells in the central nervous system (CNS) for PAS-YNS8 treated animals. Importantly, PAS-IFN8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. We next extracted RNA from spleen-derived CD4 + cells and performed high throughput qPCR for 200 IFN-response genes enriched for immune function. A striking strong correlation of gene expression and EAE clinical response was observed for two genes - CD274 (PD-L1) and CXCR3. An IFN-responsive role for PD-L1 in EAE clinical response has been recently reported elsewhere [2]. This preclinical study thus supports that we have generated a novel, potent and pharmacologically long-acting IFN-variant with potential to treat multiple sclerosis with greater efficacy than current IFN therapies available for use today. It is also provides us with a means to unravel the central IFN-response genes that are providing clinical benefit in EAE and potentially for MS.
  • Sandler N. G., Zhu R. T., Estes J. D., Boritz E., Rao S., Lifson J. D., Levin D., Schreiber G., Langer J. A. & Douek D. C. 2013. A novel type I interferon antagonist has in vivo efficacy in a model of SIV infection in rhesus macaques Cytokine. 2013 Sept , 63 (3):296-296.

    [Abstract]

    Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
  • Harari D., Abramovich R., Kallweit N., Pouly S., Zozulya-Weidenfeller A., Smith P., Schlapschy M., Koester M., Hauser H., Skerra A. & Schreiber G. 2013. Type I interferon signaling is suppressed in Experimental Autoimmune Encephalomyelitis (EAE): Implications for multiple sclerosis Cytokine. 2013 Sept , 63 (3):269-270.

    [Abstract]

    Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
  • Seidel S. A. I., Dijkman P. M., Lea W. A., van den Bogaart d. B. G., Jerabek-Willemsen M., Lazic A., Joseph J. S., Srinivasan P., Baaske P., Simeonov A., Katritch I., Melo F. A., Ladbury J. E., Schreiber G., Watts A., Braun D. & Duhr S. 2013. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions Methods. 2013 Mar , 59 (3):301-315.

    [Abstract]

    Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science. (C) 2013 Elsevier Inc. All rights reserved.
  • Apelbaum A., Yarden G., Warszawski S., Harari D. & Schreiber G. 2013. Type I Interferons Induce Apoptosis by Balancing cFLIP and Caspase-8 Independent of Death Ligands Molecular and Cellular Biology. 2013 Feb , 33 (4):800-814.

    [Abstract]

    Interferons induce a pleiotropy of responses through binding the same cell surface receptor. Here we investigated the molecular mechanism driving interferon-induced apoptosis. Using a nonbiased small interfering RNA(siRNA) screen, we show that silencing genes whose products are directly engaged in the initiation of interferon signaling completely abrogate the interferon antiproliferative response. Apoptosis-related genes such as the caspase-8, cFLIP, and DR5 genes specifically interfere with interferon-induced apoptosis, which we found to be independent of the activity of death ligands. The one gene for which silencing resulted in the strongest proapoptotic effect upon interferon signaling is the cFLIP gene, where silencing shortened the time of initiation of apoptosis from days to hours and increased dramatically the population of apoptotic cells. Thus, cFLIP serves as a regulator for interferon-induced apoptosis. A shift over time in the balance between cFLIP and caspase-8 results in downstream caspase activation and apoptosis. While gamma interferon (IFN-gamma) also causes caspase-8 upregulation, we suggest that it follows a different path to apoptosis.
  • Schreiber G. & Fleishman S. J. 2013. Computational design of protein-protein interactions Current Opinion in Structural Biology. 2013 Dec , 23 (6):903-910.

    [Abstract]

    A long-term aim of computational design is to generate specific protein protein interactions at desired affinity, specificity, and kinetics. The past three years have seen the first reports on atomically accurate de novo interactions. These were based on advances in design algorithms and the ability to harness high-throughput experimental characterization of design variants to optimize binding. Current state-of-the-art in computational design lacks precision, and therefore requires intensive experimental optimization to achieve parity with natural binders. Recent successes (and failures) point the way to future progress in design methodology that would enable routine and robust design of binders and inhibitors, while also shedding light on the essential features of biomolecular recognition.
  • Phillip Y. & Schreiber G. 2013. Formation of protein complexes in crowded environments - From in vitro to in vivo FEBS Letters. 2013 Apr , 587 (8):1046-1052.

    [Abstract]

    Traditionally, biochemical studies are performed in dilute homogenous solutions, which are very different from the dense mixture of molecules found in cells. Thus, the physiological relevance of these studies is in question. This recognition motivated scientists to formulate the effect of crowded solutions in general, and excluded volume in particular, on biochemical processes. Using polymers or proteins as crowders, it was shown that while crowding tends to significantly enhance the formation of complexes containing many subunits, dimerizations are only mildly affected. Computer simulations, together with experimental evidence, indicate soft interactions and diffusion as critical factors that operate in a concerted manner with excluded volume to modulate protein binding. Yet, these approaches do not truly mimic the cellular environment. In vivo studies may overcome this shortfall. The few studies conducted thus far suggest that in cells, binding and folding occur at rates close to those determined in dilute solutions. Obtaining quantitative biochemical information on reactions inside living cells is currently a main challenge of the field, as the complexity of the intracellular milieu was what motivated crowding research to begin with. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Phillip Y., Harel M., Khait R., Qin S., Zhou H. & Schreiber G. 2012. Contrasting Factors on the Kinetic Path to Protein Complex Formation Diminish the Effects of Crowding Agents Biophysical Journal. 2012 Sept , 103 (5):1011-1019.

    [Abstract]

    The crowded environment of cells poses a challenge for rapid protein-protein association. Yet, it has been established that the rates of association are similar in crowded and in dilute solutions. Here we probe the pathway leading to fast association between TEM1 beta-lactamase and its inhibitor protein BLIP in crowded solutions. We show that the affinity of the encounter complex, the rate of final complex formation, and the structure of the transition state are similar in crowded solutions and in buffer. The experimental results were reproduced by calculations based on the transient-complex theory for protein association. Both experiments and calculations suggest that while crowding agents decrease the diffusion constant of the associating proteins, they also induce an effective excluded-volume attraction between them. The combination of the two opposing effects thus results in nearly identical overall association rates in diluted and crowded solutions.
  • Piehler J., Thomas C., Garcia K. C. & Schreiber G. 2012. Structural and dynamic determinants of type I interferon receptor assembly and their functional interpretation Immunological Reviews. 2012 Nov , 250 :317-334.

    [Abstract]

    Type I interferons (IFNs) form a network of homologous cytokines that bind to a shared, heterodimeric cell surface receptor and engage signaling pathways that activate innate and adaptive immune responses. The ability of IFNs to mediate differential responses through the same cell surface receptor has been subject of a controversial debate and has important medical implications. During the past decade, a comprehensive insight into the structure, energetics, and dynamics of IFN recognition by its two-receptor subunits, as well as detailed correlations with their functional properties on the level of signal activation, gene expression, and biological responses were obtained. All type I IFNs bind the two-receptor subunits at the same sites and form structurally very similar ternary complexes. Differential IFN activities were found to be determined by different lifetimes and ligand affinities toward the receptor subunits, which dictate assembly and dynamics of the signaling complex in the plasma membrane. We present a simple model, which explains differential IFN activities based on rapid endocytosis of signaling complexes and negative feedback mechanisms interfering with ternary complex assembly. More insight into signaling pathways as well as endosomal signaling and trafficking will be required for a comprehensive understanding, which will eventually lead to therapeutic applications of IFNs with increased efficacy.
  • Phillip Y., Kiss V. & Schreiber G. 2012. Protein-binding dynamics imaged in a living cell Proceedings of the National Academy of Sciences of the United States of America. 2012 Jan , 109 (5):1461-1466.

    [Abstract]

    Historically, rate constants were determined in vitro and it was unknown whether they were valid for in vivo biological processes. Here, we bridge this gap by measuring binding dynamics between a pair of proteins in living HeLa cells. Binding of a beta-lactamase to its protein inhibitor was initiated by microinjection and monitored by Forster resonance energy transfer. Association rate constants for the wild-type and an electrostatically optimized mutant were only 25% and 50% lower than in vitro values, whereas no change in the rate constant was observed for a slower binding mutant. These changes are much smaller than might be anticipated considering the high macromolecular crowding within the cell. Single-cell analyses of association rate constants and fluorescence recovery after photobleaching reveals a naturally occurring variation in cell density, which is translated to an up to a twofold effect on binding rate constants. The data show that for this model protein interaction the intracellular environment had only a small effect on the association kinetics, justifying the extrapolation of in vitro data to processes in the cell.
  • Khait R. & Schreiber G. 2012. FRETex: a FRET-based, high-throughput technique to analyze protein-protein interactions PROTEIN ENGINEERING DESIGN & SELECTION. 2012 Nov , 25 (11):681-687.

    [Abstract]

    Proteinprotein interactions (PPIs) are essential for cellular viability and activity. Here, we present a rapid, semi-quantitative method (termed FRETex) to analyze PPIs, taking advantage of the strong and specific FRET signal between fused CyPET donor and YPET acceptor molecules. To demonstrate the robustness of this approach, we analyzed the interactions between three protein pairs and their muteins: TEM1-beta-lactamase binding its inhibitor BLIP, barnase binding barstar and ornithine decarboxylase binding its inhibitor antizyme. The CyPET/YPET fused proteins were produced in small quantities, and the measurements were conducted directly in the proteins crude Escherichia coli lysates without any purification step. Protein concentrations were determined from the fluorescence intensities of the lysates. While binding titration curves were produced, the resulting affinities were not always precise. Therefore, we also conducted time-resolved chase experiments using non-labeled binding partners as chasers. The acquired dissociation rate constants were in a good agreement with those measured by surface plasmon resonance. Due to the simplicity of FRETex, and the ability to obtain semi-quantitative binding data, FRETex is a suitable method for tasks such as mutant scans, protein-engineering, scanning for inhibitors and more.
  • Cohavi O., Reichmann D., Abramovich R., Tesler A. B., Bellapadrona G., Kokh D. B., Wade R. C., Vaskevich A., Rubinstein I. & Schreiber G. 2011. A Quantitative, Real-Time Assessment of Binding of Peptides and Proteins to Gold Surfaces Chemistry-A European Journal. 2011 , 17 (4):1327-1336.

    [Abstract]

    Interactions of peptides and proteins with inorganic surfaces are important to both natural and artificial systems; however, a detailed understanding of such interactions is lacking. In this study, we applied new approaches to quantitatively measure the binding of amino acids and proteins to gold surfaces. Real-time surface plasmon resonance (SPR) measurements showed that TEM1-beta-lactamase inhibitor protein (BLIP) interacts only weakly with Au nanoparticles (NPs). However, fusion of three histidine residues to BLIP (3H-BLIP) resulted in a significant increase in the binding to the Au NPs, which further increased when the histidine tail was extended to six histidines (6H-BLIP). Further increasing the number of His residues had no effect on the binding. A parallel study using continuous (111)-textured Au surfaces and single-crystalline, (111)-oriented. Au islands by ellipsometry, FTIR, and localized surface plasmon resonance (LSPR) spectroscopy further confirmed the results, validating the broad applicability of Au NPs as model surfaces. Evaluating the binding of all other natural amino acid homo-tripeptides fused to BLIP (except Cys and Pro) showed that aromatic and positively-charged residues bind preferentially to Au with respect to small aliphatic and negatively charged residues, and that the rate of association is related to the potency of binding. The binding of all fusions was irreversible. These findings were substantiated by SPR measurements of synthesized, free, soluble tripeptides using Au-NP-modified SPR chips. Here, however, the binding was reversible allowing for determination of binding affinities that correlate with the binding potencies of the related BLIP fusions. Competition assays performed between 3H-BLIP and the histidine tripeptide (3 His) suggest that Au binding residues promote the adsorption of proteins on the surface, and by this facilitate the irreversible interaction of the polypeptide chain with Au. The binding of amino acids to Au w
  • Thomas C., Moraga I., Levin D., Krutzik P. O., Podoplelova Y., Trejo A., Lee C., Yarden G., Vleck S. E., Glenn J. S., Nolan G. P., Piehler J., Schreiber G. & Garcia K. C. 2011. Structural Linkage between Ligand Discrimination and Receptor Activation by Type I Interferons Cell. 2011 Aug , 146 (4):621-632.

    [Abstract]

    Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFN alpha 2 and IFN omega reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.
  • Schreiber G. 2011. The complex relationship between interferons and their receptors dictate differential signaling Cytokine. 2011 Oct , 56 (1):22-23.

    [Abstract]

    Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
  • Lavoie T. B., Kalie E., Crisafulli-Cabatu S., Abramovich R., DiGioia G., Moolchan K., Pestka S. & Schreiber G. 2011. Binding and activity of all human alpha interferon subtypes Cytokine. 2011 Nov , 56 (2):282-289.

    [Abstract]

    Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5-5 mu M and 0.4-5 nM respectively (except for IFN-alphal - 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC(50) values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system. (C) 2011 Elsevier Ltd. All rights reserved.
  • Levin D., Harari D. & Schreiber G. 2011. Stochastic Receptor Expression Determines Cell Fate upon Interferon Treatment Molecular and Cellular Biology. 2011 Aug , 31 (16):3252-3266.

    [Abstract]

    Type I interferons trigger diverse biological effects by binding a common receptor, composed of IFNAR1 and IFNAR2. Intriguingly, while the activation of an antiviral state is common to all cells, antiproliferative activity and apoptosis affect only part of the population, even when cells are stimulated with saturating interferon concentrations. Manipulating receptor expression by different small interfering RNA (siRNA) concentrations reduced the fraction of responsive cells independent of the interferon used, including a newly generated, extremely tight-binding variant. Reduced receptor numbers increased 50% effective concentrations (EC(50)s) for alpha interferon 2 (IFN-alpha 2) but not for the tight-binding variant. A correlation between receptor numbers, STAT activation, and gene induction is observed. Our data suggest that for a given cell, the response is binary (+/-) and dependent on the stochastic expression levels of the receptors on an individual cell. A low number of receptors suffices for antiviral response and is thus a robust feature common to all cells. Conversely, a high number of receptors is required for antiproliferative activity, which allows for fine-tuning on a single-cell level.
  • Schreiber G. & Keating A. E. 2011. Protein binding specificity versus promiscuity Current Opinion in Structural Biology. 2011 Feb , 21 (1):50-61.

    [Abstract]

    Interactions between macromolecules in general, and between proteins in particular, are essential for any life process. Examples include transfer of information, inhibition or activation of function, molecular recognition as in the immune system, assembly of macromolecular structures and molecular machines, and more. Proteins interact with affinities ranging from millimolar to femtomolar and, because affinity determines the concentration required to obtain 50% binding, the amount of different complexes formed is very much related to local concentrations. Although the concentration of a specific binding partner is usually quite low in the cell (nanomolar to micromolar), the total concentration of other macromolecules is very high, allowing weak and non-specific interactions to play important roles. In this review we address the question of binding specificity, that is, how do some proteins maintain monogamous relations while others are clearly polygamous. We examine recent work that addresses the molecular and structural basis for specificity versus promiscuity. We show through examples how multiple solutions exist to achieve binding via similar interfaces and how protein specificity can be tuned using both positive and negative selection (specificity by demand). Binding of a protein to numerous partners can be promoted through variation in which residues are used for binding, conformational plasticity and/or post-translational modification. Natively unstructured regions represent the extreme case in which structure is obtained only upon binding. Many natively unstructured proteins serve as hubs in protein-protein interaction networks and such promiscuity can be of functional importance in biology.
  • Schreiber G. & Walter M. R. 2010. Cytokine-receptor interactions as drug targets Current Opinion in Chemical Biology. 2010 Aug , 14 (4):511-519.

    [Abstract]

    Cytokines are essential proteins that exert potent control over entire cell populations to fight infections and other pathologies, but can by themselves cause disease. Therefore, cytokine-related drugs act either by stimulating or blocking their activities. Our knowledge of the structures of cytokine-receptor complexes, the biophysical basis of their binding, and their mode of biological activation has substantially increased in recent years. This knowledge has been translated into new drugs and drug candidates. This review summarizes our current understanding of the receptor-mediated activity of cytokines, their relation to health and disease, and the agents in use to activate and block their actions.
  • Cohavi O., Corni S., De Rienzo R. F., Di Felice F. R., Gottschalk K. E., Hoefling M., Kokh D., Molinari E., Schreiber G., Vaskevich A. & Wade R. C. 2010. Protein-surface interactions: challenging experiments and computations Journal of Molecular Recognition. 2010 May , 23 (3):259-262.

    [Abstract]

    Protein-surface interactions are fundamental in natural processes, and have great potential for applications ranging from nanotechnology to medicine. A recent workshop highlighted the current achievements and the main challenges in the field. Copyright (C) 2009 John Wiley & Sons, Ltd.
  • Lavoie T. B., Abramovich R., Cabatu S. C., DiGioia G., Moolchan K., Pestka S. & Schreiber G. 2010. A comparison of the binding of Interferon alpha subtypes to isolated IFNAR1 and IFNAR2 with activity in antiviral and antiproliferative assays Cytokine. 2010 Oct , 52 (2-Jan):86-86.

    [Abstract]

    Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
  • Harari D., Levin D., Abramovich R. & Schreiber G. 2010. Reduced interferon receptor levels resulting from either knockdown or prolonged Type I interferon treatment promote a shift in cellular responsiveness Cytokine. 2010 Oct , 52 (2-Jan):57-58.

    [Abstract]

    Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
  • Potapov V., Cohen M., Inbar Y. & Schreiber G. 2010. Protein structure modelling and evaluation based on a 4-distance description of side-chain interactions BMC Bioinformatics. 2010 Jul , 11 .

    [Abstract]

    Background: Accurate evaluation and modelling of residue-residue interactions within and between proteins is a key aspect of computational structure prediction including homology modelling, protein-protein docking, refinement of low-resolution structures, and computational protein design. Results: Here we introduce a method for accurate protein structure modelling and evaluation based on a novel 4-distance description of residue-residue interaction geometry. Statistical 4-distance preferences were extracted from high-resolution protein structures and were used as a basis for a knowledge-based potential, called Hunter. We demonstrate that 4-distance description of side chain interactions can be used reliably to discriminate the native structure from a set of decoys. Hunter ranked the native structure as the top one in 217 out of 220 high-resolution decoy sets, in 25 out of 28 "Decoys 'R' Us" decoy sets and in 24 out of 27 high-resolution CASP7/8 decoy sets. The same concept was applied to side chain modelling in protein structures. On a set of very high-resolution protein structures the average RMSD was 1.47 angstrom for all residues and 0.73 angstrom for buried residues, which is in the range of attainable accuracy for a model. Finally, we show that Hunter performs as good or better than other top methods in homology modelling based on results from the CASP7 experiment. The supporting web site http://bioinfo.weizmann.ac.il/hunter/ was developed to enable the use of Hunter and for visualization and interactive exploration of 4-distance distributions. Conclusions: Our results suggest that Hunter can be used as a tool for evaluation and for accurate modelling of residue-residue interactions in protein structures. The same methodology is applicable to other areas involving high-resolution modelling of biomolecules.
  • Cohavi O., Tobi D. & Schreiber G. 2009. Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data Journal of Molecular Biology. 2009 Jul , 390 (3):503-515.

    [Abstract]

    Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed. (C) 2009 Elsevier Ltd. All rights reserved.
  • Cohen M., Potapov V. & Schreiber G. 2009. Four Distances between Pairs of Amino Acids Provide a Precise Description of their Interaction PLoS Computational Biology. 2009 Aug , 5 (8).

    [Abstract]

    The three-dimensional structures of proteins are stabilized by the interactions between amino acid residues. Here we report a method where four distances are calculated between any two side chains to provide an exact spatial definition of their bonds. The data were binned into a four-dimensional grid and compared to a random model, from which the preference for specific four-distances was calculated. A clear relation between the quality of the experimental data and the tightness of the distance distribution was observed, with crystal structure data providing far tighter distance distributions than NMR data. Since the four-distance data have higher information content than classical bond descriptions, we were able to identify many unique inter-residue features not found previously in proteins. For example, we found that the side chains of Arg, Glu, Val and Leu are not symmetrical in respect to the interactions of their head groups. The described method may be developed into a function, which computationally models accurately protein structures.
  • Potapov V., Cohen M. & Schreiber G. 2009. Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details PROTEIN ENGINEERING DESIGN & SELECTION. 2009 Sept , 22 (9):553-560.

    [Abstract]

    Methods for protein modeling and design advanced rapidly in recent years. At the heart of these computational methods is an energy function that calculates the free energy of the system. Many of these functions were also developed to estimate the consequence of mutation on protein stability or binding affinity. In the current study, we chose six different methods that were previously reported as being able to predict the change in protein stability (delta delta G) upon mutation: CC/PBSA, EGAD, FoldX, I-Mutant2.0, Rosetta and Hunter. We evaluated their performance on a large set of 2156 single mutations, avoiding for each program the mutations used for training. The correlation coefficients between experimental and predicted delta delta G values were in the range of 0.59 for the best and 0.26 for the worst performing method. All the tested computational methods showed a correct trend in their predictions, but failed in providing the precise values. This is not due to lack in precision of the experimental data, which showed a correlation coefficient of 0.86 between different measurements. Combining the methods did not significantly improve prediction accuracy compared to a single method. These results suggest that there is still room for improvement, which is crucial if we want forcefields to perform better in their various tasks.
  • Moraga I., Harari D., Schreiber G., Uze G. & Pellegrini S. 2009. Receptor Density Is Key to the Alpha2/Beta Interferon Differential Activities Molecular and Cellular Biology. 2009 Sept , 29 (17):4778-4787.

    [Abstract]

    Multiple type I interferons (IFN-alpha/beta) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-alpha/beta differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype- specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-beta was more potent than IFN-alpha 2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha 2 matched or even surpassed IFN-beta in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.
  • Schreiber G., Harari D., Kalie E. & Jaitin D. A. 2009. Designer interferons reveal the relation between receptor binding and differential biological activity Cytokine. 2009 Oct , 48 (2-Jan):121-121.

    [Abstract]

    Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology
  • Phillip Y., Sherman E., Haran G. & Schreiber G. 2009. Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions Biophysical Journal. 2009 Aug , 97 (3):875-885.

    [Abstract]

    Studies of protein-protein interactions, carried out in polymer solutions, are designed to mimic the crowded environment inside living cells. It was shown that crowding enhances oligomerization and polymerization of macromolecules. Conversely, we have shown that crowding has only a small effect on the rate of association of protein complexes. Here, we investigated the equilibrium effects of crowding on protein heterodimerization of TEM1-beta-lactamase with,Q-lactamase inhibitor protein (BLIP) and barnase with barstar. We also contrasted these with the effect of crowding on the weak binding pair CyPet-YPet. We measured the association and dissociation rates as well as the affinities and thermodynamic parameters of these interactions in polyethylene glycol and dextran solutions. For TEM1-BLIP and for barnase-barstar, only a minor reduction in association rate constants compared to that expected based on solution viscosity was found. Dissociation rate constants showed similar levels of reduction. Overall, this resulted in a binding affinity that is quite similar to that in aqueous solutions. On the other hand, for the CyPet-YPet pair, aggregation, and not enhanced dimerization, was detected in polyethylene glycol solutions. The results suggest that typical crowding agents have only a small effect on specific protein-protein dimerization reactions. Although crowding in the cell results from proteins and other macromolecules, one may still speculate that binding in vivo is not very different from that measured in dilute solutions.
  • Schreiber G., Haran G. & Zhou H. 2009. Fundamental Aspects of Protein-Protein Association Kinetics Chemical Reviews. 2009 Mar , 109 (3):839-860.
  • Kiel C., Filchtinski D., Spoerner M., Schreiber G., Kalbitzer H. R. & Herrmann C. 2009. Improved Binding of Raf to Ras.GDP Is Correlated with Biological Activity Journal of Biological Chemistry. 2009 Nov , 284 (46):31893-31902.

    [Abstract]

    The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras.Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras.GTP versus Ras.GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.
  • Rahat O., Alon U., Levy Y. & Schreiber G. 2009. Understanding hydrogen-bond patterns in proteins using network motifs Bioinformatics. 2009 Nov , 25 (22):2921-2928.

    [Abstract]

    Protein structures can be viewed as networks of contacts (edges) between amino-acid residues (nodes). Here we dissect proteins into sub-graphs consisting of six nodes and their corresponding edges, with an edge being either a backbone hydrogen bond (H-bond) or a covalent interaction. Six thousand three hundred and twenty-two such sub-graphs were found in a large non-redundant dataset of high-resolution structures, from which 35 occur much more frequently than in a random model. Many of these significant sub-graphs (also called network motifs) correspond to sub-structures of a helices and beta-sheets, as expected. However, others correspond to more exotic sub-structures such as 3(10) helix, Schellman motif and motifs that were not defined previously. This topological characterization of patterns is very useful for producing a detailed differences map to compare protein structures. Here we analyzed in details the differences between NMR, molecular dynamics (MD) simulations and X-ray structures for Lysozyme, SH3 and the lambda repressor. In these cases, the same structures solved by NMR and simulated by MD showed small but consistent differences in their motif composition from the crystal structures, despite a very small root mean square deviation (RMSD) between them. This may be due to differences in the pair-wise energy functions used and the dynamic nature of these proteins.
  • Harel M., Spaar A. & Schreiber G. 2009. Fruitful and Futile Encounters along the Association Reaction between Proteins Biophysical Journal. 2009 May , 96 (10):4237-4248.

    [Abstract]

    The association reaction between pairs of proteins proceeds through an encounter complex that develops into the final complex. Here, we combined Brownian dynamics simulations with experimental studies to analyze the structures of the encounter complexes along the association reaction between TEM1-beta-lactamase and its inhibitor, beta-lactamase-inhibitor protein. The encounter complex can be considered as an ensemble of short-lived low free-energy states that are stabilized primarily by electrostatic forces and desolvation. For the wild-type, the simulation showed two main encounter regions located outside the physical binding site. One of these regions was located near the experimentally determined transition state. To validate whether these encounters are fruitful or futile, we examined three groups of mutations that altered the encounter. The first group consisted of mutations that increased the experimental rate of association through electrostatic optimization. This resulted in an increase in the size of the encounter region located near the experimentally determined transition state, as well as a decrease in the energy of this region and an increase in the number of successful trajectories (i.e., encounters that develop into complex). A second group of mutations was specifically designed to either increase or decrease the size and energy of the second encounter complex, but either way it did not affect k(on). A third group of mutations consisted of residues that increased k(on) without significantly affecting the encounter complexes. These results indicate that the size and energy of the encounter regions are only two of several parameters that lead to fruitful association, and that electrostatic optimization is a major driving force in fast association.
  • Pan M., Kalie E., Scaglione B. J., Raveche E. S., Schreiber G. & Langer J. A. 2008. Mutation of the IFNAR-1 Receptor Binding Site of Human IFN-alpha 2 Generates Type I IFN Competitive Antagonists Biochemistry. 2008 Nov , 47 (46):12018-12027.

    [Abstract]

    Type I interferons (IFNs) are multifunctional cytokines that activate cellular responses by binding a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Although the binding of IFNs to IFNAR-2 is well characterized, the binding to the lower affinity IFNAR-1 remains less well understood. Previous reports identified a region of human IFN-alpha 2 on the B and C helices ("site 1A": N65, L80, Y85, Y89) that plays a key role in binding IFNAR-1 and contributes strongly to differential activation by various type I IFNs. The current studies demonstrate that residues on the D helix are also involved in IFNAR-1 binding. In particular, residue 120 (Arg in IFN-alpha 2; Lys in IFN-alpha 2/alpha 1)appears to be a "hot-spot" residue: substitution by alanine significantly decreased biological activity, and the charge-reversal mutation of residue 120 to Glu caused drastic loss of antiviral and antiproliferative activity for both IFN-alpha 2 and IFN-alpha 2/alpha 1. Mutations in residues of helix D maintained their affinity for IFNAR-2 but had decreased affinity for IFNAR-1. Single-site or multiple-site mutants in the IFNAR-1 binding site that had little or no detectable in vitro biological activity were capable of blocking in vitro antiviral and antiproliferative activity of native IFN-alpha 2; i.e., they are type I IFN antagonists. These prototype IFN antagonists can be developed further for possible therapeutic use in systemic lupus erythematosus, and analogous molecules can be designed for use in animal models.
  • Rahat O., Yitzhaky A. & Schreiber G. 2008. Cluster conservation as a novel tool for studying protein-protein interactions evolution Proteins-Structure Function And Bioinformatics. 2008 May , 71 (2):621-630.

    [Abstract]

    Protein-protein interactions networks has come to be a buzzword associated with nets containing edges that represent a pair of interacting proteins (e.g. hormone-receptor, enzyme-inhibitor, antigen-antibody, and a subset of multichain biological machines). Yet, each such interaction composes its own unique network, in which vertices represent amino acid residues, and edges represent atomic contacts. Recent studies have shown that analyses of the data encapsulated in these detailed networks may impact predictions of structure-function correlation. Here, we study homologous families of protein-protein interfaces, which share the same fold but vary in sequence. In this context, we address what properties of the network are shared among relatives with different sequences (and hence different atomic interactions) and which are not. Herein, we develop the general mathematical framework needed to compare the modularity of homologous networks. We then apply this analysis to the structural data of a few interface families, including hemoglobin alpha-beta, growth hormone-receptor, and Serine protease-inhibitor. Our results suggest that interface modularity is an evolutionarily conserved property. Hence, protein-protein interfaces can be clustered down to a few modules, with the boundaries being evolutionarily conserved along homologous complexes. This suggests that protein engineering of protein-protein binding sites may be simplified by varying each module, but retaining the overall modularity of the interface.
  • Reichmann D., Phillip Y., Carmi A. & Schreiber G. 2008. On the contribution of water-mediated interactions to protein-complex stability Biochemistry. 2008 Jan , 47 (3):1051-1060.

    [Abstract]

    Protein-water interactions have long been recognized as a major determinant of chain folding, conformational stability, binding specificity and catalysis. However, the detailed effects of water on stabilizing protein-protein interactions remain elusive. A way to test experimentally the contribution of water-mediated interactions is by applying double mutant cycle analysis on pairs of residues that do not form direct interactions, but are bridged by water. Seven such interactions within the interface between TEM1 and BLIP proteins were evaluated. No significant interaction free energy was found between either of them. Water can bridge interactions, but also stabilize the structure of the monomer. To distinguish between these, we performed a bioinformatic analysis using AQUAPROT (http://bioinfo.weizmann.ac.il/ aquaprot) to determine the degree of water conservation between the bound and unbound states. 29 structures of twelve complexes and 20 related monomers were analyzed. Of the 262 water molecules located within the interfaces, 145 were conserved between the unbound and bound structures. Strikingly, all 50 buried or partially buried waters in the monomer structures were conserved at the same location in the bound structures. Thus, buried waters have an important role in stabilizing the monomer fold rather than contributing to protein-protein binding, and are not replaced by residues from the incoming protein. Taking together the experimental and bioinformatics evidence suggests that exposed waters within the interface may be good sites for protein engineering, while buried or mostly buried waters should be left unchanged.
  • Cohen M., Reichmann D., Neuvirth H. & Schreiber G. 2008. Similar chemistry, but different bond preferences in inter versus intra-protein interactions Proteins-Structure Function And Bioinformatics. 2008 Aug , 72 (2):741-753.

    [Abstract]

    Proteins fold into a well-defined structure as a result of the collapse of the polypeptide chain, while transient protein-complex formation mainly is a result of binding of two folded individual monomers. Therefore, a protein-protein interface does not resemble the core of monomeric proteins, but has a more polar nature. Here, we address the question of whether the physico-chemical characteristics of intraprotein versus interprotein bonds differ, or whether interfaces are different from folded monomers only in the preference for certain types of interactions. To address this question we assembled a high resolution, nonredundant, protein-protein interaction database consisting of 1374 homodimer and 572 heterodimer complexes, and compared the physico-chemical properties of these interactions between protein interfaces and monomers. We performed extensive statistical analysis of geometrical properties of interatomic interactions of different types: hydrogen bonds, electrostatic interactions, and aromatic interactions. Our study clearly shows that there is no significant difference in the chemistry, geometry, or packing density of individual interactions between interfaces and monomeric structures. However, the distribution of different bonds differs. For example, side-chain-side-chain interactions constitute over 62% of all interprotein interactions, while they make up only 36% of the bonds stabilizing a protein structure. As on average, properties of backbone interactions are different from those of side chains, a quantitative difference is observed. Our findings clearly show that the same knowledge-based potential can be used for protein-binding sites as for protein structures. However, one has to keep in mind the different architecture of the interfaces and their unique bond preference.
  • Kalie E., Jaitin D. A., Podoplelova Y., Piehler J. & Schreiber G. 2008. The Stability of the Ternary Interferon-Receptor Complex Rather than the Affinity to the Individual Subunits Dictates Differential Biological Activities Journal of Biological Chemistry. 2008 Nov , 283 (47):32925-32936.

    [Abstract]

    Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFN alpha 2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC50 values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFN alpha 2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.
  • Bravman T., Bronner V., Nahshol O. & Schreiber G. 2008. The ProteOn XPR36 (TM) Array System-High Throughput Kinetic Binding Analysis of Biomolecular Interactions Cellular and Molecular Bioengineering. 2008 Dec , 1 (4):216-228.

    [Abstract]

    Surface plasmon resonance (SPR) technology is a central, widely used tool for kinetic studies of interactions between unlabeled biomolecules in real time. The ProteOn XPR36 protein interaction array system possesses all the qualities of high-level SPR biosensing technology combined with high-throughput and multiplexing capabilities. This system is based on the built-in orientation controlled multi-channel module of the ProteOn XPR36 system, which allows parallel measurement of multiple binding interactions between as many as six protein pairs. This kind of multiplexing is done efficiently on the ProteOn XPR36 system using the innovative technique of One-shot Kinetics (TM), which allows simultaneous monitoring of multiple protein pair interactions. Here we demonstrate the capabilities of the ProteOn XPR36 for the measurements of a wide range of biomolecules interactions including proteins, DNA oligos, small molecules and antibodies and discuss its optimal mode of use.
  • Potapov V., Reichmann D., Abramovich R., Filchtinski D., Zohar N., Ben Halevy H. D., Edelman M., Sobolev V. & Schreiber G. 2008. Computational Redesign of a Protein-Protein Interface for High Affinity and Binding Specificity Using Modular Architecture and Naturally Occurring Template Fragments Journal of Molecular Biology. 2008 Dec , 384 (1):109-119.

    [Abstract]

    A new method is presented for the redesign of protein-protein interfaces, resulting in specificity of the designed pair while maintaining high affinity. The design is based on modular interface architecture and was carried out on the interaction between TEM1 beta-lactamase and its inhibitor protein, beta-lactamase inhibitor protein. The interface between these two proteins is composed of several mostly independent modules. We previously showed that it is possible to delete a complete module without affecting the overall structure of the interface. Here, we replace a complete module with structure fragments taken from nonrelated proteins. Nature-optimized fragments were chosen from 10(7) starting templates found in the Protein Data Bank. A procedure was then developed to identify sets of interacting template residues with a backbone arrangement mimicking the original module. This generated a final list of 361 putative replacement modules that were ranked using a novel scoring function based on grouped atom-atom contact surface areas. The top-ranked designed complex exhibited an affinity of at least the wild-type level and a mode of binding that was remarkably specific despite the absence of negative design in the procedure. In retrospect, the combined application of three factors led to the success of the design approach: utilizing the modular construction of the interface, capitalizing on native rather than artificial templates, and ranking with an accurate atom-atom contact surface scoring function. (c) 2008 Elsevier Ltd. All rights reserved.
  • Adam Y., Tayer N., Rotem D., Schreiber G. & Schuldiner S. 2007. The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter Proceedings of the National Academy of Sciences of the United States of America. 2007 Nov , 104 (46):17989-17994.

    [Abstract]

    EmrE is an Escherichia coli H+-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP+, a high-affinity substrate, is 2 x 10(7) M-1 center dot s(-1), a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the k(off) of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.
  • Hodis E., Schreiber G., Rother K. & Sussman J. 2007. eMovie: a storyboard-based tool for making molecular movies Trends in Biochemical Sciences. 2007 May , 32 (5):199-204.

    [Abstract]

    The 3D structures of macromolecules are difficult to grasp and also to communicate. By their nature, movies or animations are particularly useful for highlighting key features by offering a 'guided tour' of structures and conformation changes. However, high-quality movies are rarely seen because they are currently difficult and time consuming to make. By adopting the traditional movie 'storyboard' concept, which gives guidance and direction to filming, eMovie makes the creation of lengthy molecular animations much easier. This tool is a plug-in for the open-source molecular graphics program PyMOL, and enables experts and novices alike to produce informative and high-quality molecular animations.
  • Kozer N., Kuttner Y. Y., Haran G. & Schreiber G. 2007. Protein-protein association in polymer solutions: From dilute to semidilute to concentrated Biophysical Journal. 2007 Mar , 92 (6):2139-2149.

    [Abstract]

    In a typical cell, proteins function in the crowded cytoplasmic environment where 30% of the space is occupied by macromolecules of varying size and nature. This environment may be simulated in vitro using synthetic polymers. Here, we followed the association and diffusion rates of TEM1-beta-lactamase (TEM) and the beta-lactamase inhibitor protein (BLIP) in the presence of crowding agents of varying molecular mass, from monomers (ethylene glycol, glycerol, or sucrose) to polymeric agents such as different polyethylene glycols (PEGs, 0.2-8 kDa) and Ficoll. An inverse linear relation was found between translational diffusion of the proteins and viscosity in all solutions tested, in accordance with the Stokes-Einstein (SE) relation. Conversely, no simple relation was found between either rotational diffusion rates or association rates (kon) and viscosity. To assess the translational diffusion-independent steps along the association pathway, we introduced a new factor, alpha, which corrects the relative change in kon by the relative change in solution viscosity, thus measuring the deviations of the association rates from SE behavior. We found that these deviations were related to the three regimes of polymer solutions: dilute, semidilute, and concentrated. In the dilute regime PEGs interfere with TEM-BLIP association by introducing a repulsive force due to solvophobic preferential hydration, which results in slower association than predicted by the SE relation. Crossing over from the dilute to the semidilute regime results in positive deviations from SE behavior, i.e., relatively faster association rates. These can be attributed to the depletion interaction, which results in an effective attraction between the two proteins, winning over the repulsive force. In the concentrated regime, PEGs again dramatically slow down the association between TEM and BLIP, an effect that does not depend on the physical dimensions of PEGs, but rather on their mass concentration. This i
  • Reichel A., Schaible D., Al Furoukh F. N., Cohen M., Schreiber G. & Piehler J. 2007. Noncovalent, site-specific biotinylation of histidine-tagged proteins Analytical Chemistry. 2007 Nov , 79 (22):8590-8600.

    [Abstract]

    Site-specific conjugation of proteins to surfaces, spectroscopic probes, or other functional units is a key task for implementing biochemical assays. The streptavidin-biotin interaction has proven a highly versatile tool for detection, quantification, and functional analysis of proteins. We have developed an approach for site-specific reversible biotinylation of recombinant proteins through their histidine tag using biotin conjugated to the multivalent chelator trisnitrilotriacetic acid ((BT)tris-NTA). Stable binding of BTtris-NTA to His-tagged proteins was demonstrated, which is readily reversed by addition of imidazole, enabling versatile conjugation schemes in solution as well as at interfaces. Gel filtration experiments revealed that His-tagged proteins bind to streptavidin doped with (BT)tris-NTA in a 2:1 stoichiometry. Furthermore, an increased binding affinity toward His-tagged proteins was observed for BTtris-NTA linked to streptavidin compared to tris-NTA in solution and on surfaces. These results indicate an efficient cooperative interaction of two adjacent tris-NTA moieties with a single His-tag, yielding an extremely tight complex with a lifetime of several days. We demonstrate several applications of BTtris-NTA including multiplexed capturing of proteins to biosensor surfaces, cell surface labeling, and Western blot detection. The remarkable selectivity of the His-tag-specific biotinylation, as well as the highly stable, yet reversible complex provides the basis for numerous further applications for functional protein analysis.
  • Reichmann D., Cohen M., Abramovich R., Dym O., Lim D., Strynadka N. C. J. & Schreiber G. 2007. Binding hot spots in the TEM1-BLIP interface in light of its modular architecture Journal of Molecular Biology. 2007 Jan , 365 (3):663-679.

    [Abstract]

    Proteins bind one another in aqua's solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues' where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 beta-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein-protein binding occur via the structural reorganization of the binding modules, either by a "lock and key" or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high-resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design. (c) 2006 Elsevier Ltd. All rights reserved.
  • Uze G., Schreiber G., Piehler J. & Pellegrini S. 2007. The receptor of the type I interferon family Interferon: The 50Th Anniversary. 2007 , 316 :71-95.

    [Abstract]

    All type I IFNs act through a single cell surface receptor composed of the IFNAR1 and IFNAR2 subunits and two associated cytoplasmic tyrosine kinases of the Janus family, Tyk2 and Jak1. A central issue in type I IFN biology is to understand how 1 a multitude of subtypes can generate similar signaling outputs but also govern specific 1 cellular responses. This review summarizes results from the last decade that contributed to our current state of knowledge of IFN-receptor complex structure and assembly.
  • Jaitin D. A. & Schreiber G. 2007. Upregulation of a small subset of genes drives type I interferon-induced antiviral memory Journal of Interferon and Cytokine Research. 2007 Aug , 27 (8):653-664.

    [Abstract]

    Interferons ( IFNs) stand in the frontline of defense against viral infections. In this study, we aimed at characterizing the gene expression profile specific to the antiviral effect out of the hundreds of genes involved also in other IFN activities. We found that the IFN- induced antiviral state is maintained for a prolonged time even after IFN occlusion. This was achieved through the active expression of a small set of <40 genes long after IFN was occluded, from which two groups are distinguished: one includes genes participating in direct inhibition of viral replication, such as Mx and OAS; the second group is related to antigen presentation, including all genes involved in the proteasome-to-immunoproteasome switch and class I MHC genes. Transcription of these genes continued after IFN removal and was Stat1 independent, suggesting the involvement of other signaling elements in addition to the canonical signal transduction pathway. Not less important were genes whose upregulation, in cases by many fold, is terminated once IFN is removed. Among these are viral sensing genes, such as retinoic acid-inducible gene-I protein (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and toll-like receptor (TLR), cytokines, and apoptotic-related genes. Our findings provide a systemwide depiction of prolonged intracellular antiviral protection without the need for ongoing IFN stimulation.
  • Kalie E., Jaitin D. A., Abramovich R. & Schreiber G. 2007. An interferon alpha 2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities Journal of Biological Chemistry. 2007 Apr , 282 (15):11602-11611.

    [Abstract]

    All alpha-interferons ( IFN alpha) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN alpha 2. Here, we constructed a phage display library by randomizing three positions on IFN alpha 2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S ( YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFN alpha 2, and 3-fold tighter compared with IFN beta. Binding of YNS to IFNAR2 was comparable with wild-type IFN alpha 2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN alpha 2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFN alpha 2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFN alpha 2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency.
  • Reichmann D., Rahat O., Cohen M., Neuvirth H. & Schreiber G. 2007. The molecular architecture of protein-protein binding sites Current Opinion in Structural Biology. 2007 Feb , 17 (1):67-76.

    [Abstract]

    The formation of specific protein interactions plays a crucial role in most, if not all, biological processes, including signal transduction, cell regulation, the immune response and others. Recent advances in our understanding of the molecular architecture of protein-protein binding sites, which facilitates such diversity in binding affinity and specificity, are enabling us to address key questions. What is the amino acid composition of binding sites? What are interface hotspots? How are binding sites organized? What are the differences between tight and weak interacting complexes? How does water contribute to binding? Can the knowledge gained be translated into protein design? And does a universal code for binding exist, or is it the architecture and chemistry of the interface that enable diverse but specific binding solutions?
  • Neuvirth H., Heinemann U., Birnbaum D., Tishby N. & Schreiber G. 2007. ProMateus: an open research approach to protein-binding sites analysis Nucleic Acids Research. 2007 Jul , 35 (suppl 2):W543-W548.

    [Abstract]

    The development of bioinformatic tools by individual labs results in the abundance of parallel programs for the same task. For example, identification of binding site regions between interacting proteins is done using: ProMate, WHISCY, PPI-Pred, PINUP and others. All servers first identify unique properties of binding sites and then incorporate them into a predictor. Obviously, the resulting prediction would improve if the most suitable parameters from each of those predictors would be incorporated into one server. However, because of the variation in methods and databases, this is currently not feasible. Here, the protein-binding site prediction server is extended into a general protein-binding sites research tool, ProMateus. This web tool, based on ProMate's infrastructure enables the easy exploration and incorporation of new features and databases by the user, providing an evaluation of the benefit of individual features and their combination within a set framework. This transforms the individual research into a community exercise, bringing out the best from all users for optimized predictions. The analysis is demonstrated on a database of protein protein and protein-DNA interactions. This approach is basically different from that used in generating meta-servers. The implications of the open-research approach are discussed. ProMateus is available at http://bip.weizmann.ac.il/promate.
  • Harel M., Cohen M. & Schreiber G. 2007. On the dynamic nature of the transition state for protein-protein association as determined by double-mutant cycle analysis and simulation Journal of Molecular Biology. 2007 Aug , 371 (1):180-196.

    [Abstract]

    The process of protein-protein association starts with their random collision, which may develop into an encounter complex followed by a transition state and final complex formation. Here we aim to experimentally characterize the nature of the transition state of protein-protein association for three different protein-protein interactions; Barnase-Barstar, TEM1-BLIP and IFN alpha 2-IFNAR2, and use the data to model the transition state structures. To model the transition state, we determined inter-protein distance-constraints of the activated complex by using double mutant cycles (DMC) assuming that interacting residues are spatially close. Significant Delta Delta G double dagger(int) values were obtained only between residues on Barnase and Barstar. However, introducing specific mutations that optimize the charge complementarity between BLIP and TEM1 resulted in the introduction of significant Delta Delta G double dagger(int) values also between residues of these two proteins. While electrostatic interactions make major contributions towards stabilizing the transition state, we show two examples where steric hindrance exerts an effect on the transition state as well. To model the transition-state structures from the experimental Delta Delta G double dagger(int) values, we introduced a method for structure perturbation, searching for those inter-protein orientations that best support the experimental Delta Delta G double dagger(int) values. Two types of transition states were found, specific as observed for Barnase-Barstar and the electrostatically optimized TEM1-BLIP mutants, and diffusive as shown for wild-type TEM1-BLIP and IFN(alpha 2-IFNAR2. The specific transition states are characterized by defined inter-protein orientations, which cannot be modeled for the diffusive transition states. Mutations introduced through rational design can change the transition state from diffusive to specific. Together, these data provide a structural view of the mechanism allo
  • Raveh B., Rahat O., Basri R. & Schreiber G. 2007. Rediscovering secondary structures as network motifs - an unsupervised learning approach Bioinformatics. 2007 Jan , 23 (2):E163-E169.

    [Abstract]

    Motivation: Secondary structures are key descriptors of a protein fold and its topology. In recent years, they facilitated intensive computational tasks for finding structural homologues, fold prediction and protein design. Their popularity stems from an appealing regularity in patterns of geometry and chemistry. However, the definition of secondary structures is of subjective nature. An unsupervised de-novo discovery of these structures would shed light on their nature, and improve the way we use these structures in algorithms of structural bioinformatics. Methods: We developed a new method for unsupervised partitioning of undirected graphs, based on patterns of small recurring network motifs. Our input was the network of all H-bonds and covalent interactions of protein backbones. This method can be also used for other biological and non-biological networks. Results: In a fully unsupervised manner, and without assuming any explicit prior knowledge, we were able to rediscover the existence of conventional alpha-helices, parallel beta-sheets, anti-parallel sheets and loops, as well as various non-conventional hybrid structures. The relation between connectivity and crystallographic temperature factors establishes the existence of novel secondary structures.
  • Schmeisser H., Kontsek P., Esposito D., Gillette W., Schreiber G. & Zoon K. C. 2006. Binding characteristics of IFN-alpha subvariants to IFNAR2-EC and influence of the 6-histidine tag Journal of Interferon and Cytokine Research. 2006 Dec , 26 (12):866-876.

    [Abstract]

    The expression, purification, detection, and assay of recombinant proteins have been made more convenient and rapid by the use of small affinity tags. To facilitate the purification of interferon-alpha 2c ( IFN-alpha 2c) by metal chelate affinity chromatography, N-terminal 6-histidine tag was introduced via genetic manipulation. Two preparations of IFN material were purified; one contained IFN-alpha 2c with the 6-histidine tag, and the other contained IFN-alpha 2c without the 6-histidine tag. The antigenic properties of the human IFN-alpha 2c subvariant with and without the 6-histidine tag, as well as the effects of the N-terminal 6-histidine tag on IFN-alpha 2c interaction with the extracellular domain of human IFN-alpha receptor chain 2 ( IFNAR2-EC) were examined. For the purposes of this study, IFNs were characterized by Western blots with anti-IFN monoclonal antibodies ( mAb) and bioassays. Immunoblot analyses showed differences between IFN-alpha 2c-6-histidine tag and IFN-alpha 2a, b, c in their interaction with IFNAR2-EC. We also observed differences between IFN-alpha 2c-6-histidine tag and IFN-alpha 2a, b, c in bioactivities. This study is the first report that shows that an N-terminal 6-histidine tag on IFN-alpha 2c can affect its interaction with receptor and cause a different bioactivity.
  • Slutzki M., Jaitin D. A., Ben-Yehezkel T. & Schreiber G. 2006. Variations in the unstructured C-terminal tail of interferons contribute to differential receptor binding and biological activity Journal of Molecular Biology. 2006 Jul , 360 (5):1019-1030.

    [Abstract]

    Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFN alpha s are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of lFN alpha 2 with that of IFN alpha 8 and IFN beta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of lFN alpha 8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK; second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results, we suggest that the unstructured tail of IFN alpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2. (c) 2006 Elsevier Ltd. All rights reserved.
  • Jaitin D., Roisman L., Jaks E., Gavutis M., Pichler J., Van der Heyden d. H. J., Uze G. & Schreiber G. 2006. Inquiring into the differential action of interferons (IFNs): an IFN-alpha 2 mutant with enhanced affinity to IFNAR1 is functionally similar to IFN-beta Molecular and Cellular Biology. 2006 Mar , 26 (5):1888-1897.

    [Abstract]

    Alpha and beta interferons (IFN-alpha and IFN-beta) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-alpha 2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-beta. Compared to wild-type (wt) IFN-a2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-beta, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-beta, promotes a differentially higher anti proliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-beta and higher levels of gene induction or repression than those for wt IFN-alpha 2. Thus, we show that the differential activities of IFN-beta are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-alpha subtypes suggests that IFN-alpha has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-beta.
  • Schreiber G. & Serrano L. 2005. Folding and binding: an extended family business - Editorial overview Current Opinion in Structural Biology. 2005 Feb , 15 (1):1-3.
  • Albeck S., Burstein Y., Dym O., Jacobovitch Y., Levi N., Meged R., Michael Y., Peleg Y., Prilusky J., Schreiber G., Silman I., Unger T. & Sussman J. 2005. Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC) Acta Crystallographica Section D-Biological Crystallography. 2005 Oct , 61 :1364-1372.

    [Abstract]

    The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.
  • Schreiber G. 2005. The Nitty-Gritty of protein interactions Structure. 2005 Dec , 13 (12):1737-1738.
  • Kuttner Y., Kozer N., Segal E., Schreiber G. & Haran G. 2005. Separating the contribution of translational and rotational diffusion to protein association Journal of the American Chemical Society. 2005 Nov , 127 (43):15138-15144.

    [Abstract]

    The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and P-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.
  • Reichmann D., Rahat O., Albeck S., Meged R., Dym O. & Schreiber G. 2005. The modular architecture of protein-protein binding interfaces Proceedings of the National Academy of Sciences of the United States of America. 2005 Jan , 102 (1):57-62.

    [Abstract]

    Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved.
  • Roisman L., Jaitin D., Baker D. & Schreiber G. 2005. Mutational analysis of the IFNAR1 binding site on IFN alpha 2 reveals the architecture of a weak ligand-receptor binding-site Journal of Molecular Biology. 2005 Oct , 353 (2):271-281.

    [Abstract]

    Type I interferons activate cellular responses by forming a ternary complex with two receptor components, IFNAR1 and IFNAR2. While the binding of the IFNAR2 receptor to interferon is of high affinity and well characterized, the binding to IFNAR1 is weak, transient, and poorly understood. Here, we mapped the complete binding region of IFNAR1 on IFN alpha 2 by creating a panel of 21 single alanine mutant proteins, and determined t affinities. The IFNAR1 binding site on IFN alpha 2 maps to the center of the B and C helices, opposite to the binding site for IFNAR2. No hot spots for binding were found in the interface, with individual mutations having an up to fivefold effect on binding. Of the nine residues that affected binding, three adjacent conserved residues, located on the B helix, conferred an increase in the binding affinity to IFNARI, as well as an increase in the biological activity of the interferon mutant. This suggests that binding of alpha interferons to the IFNAR1 receptor is sub-optimal. A correlation between binding affinity and biological activity was found, albeit not across the whole range of affinities. In WISH cells, but not DAUDI cells, the anti-proliferative activity was markedly affected by fluctuations in the IFN alpha 2 affinity towards the IFNAR1 receptor. On the other hand, the antiviral activity of interferons on WISH cells seems to change in accordance to the binding affinity towards IFNAR1 only as long as the binding affinity is not beyond twofold of the wild-type. In accordance, the biological roles of the two interferon-receptor subunits are discussed. (c) 2005 Elsevier Ltd. All rights reserved.
  • Shaul Y. & Schreiber G. 2005. Exploring the charge space of protein-protein association: A proteomic study Proteins-Structure Function And Bioinformatics. 2005 Aug , 60 (3):341-352.

    [Abstract]

    The rate of association of a protein complex is a function of an intrinsic basal rate and of the magnitude of electrostatic steering. In the present study we analyze the contribution of electrostatics towards the association rate of proteins in a database of 68 transient hetero-protein-protein complexes. Our calculations are based on an upgraded version of the computer algorithm PARE, which was shown to successfully predict the impact of mutations on k(on) by calculating the difference in Columbic energy of interaction of a pair of proteins. HyPare (http://bip.weizmann.ac.il/HyPare), automatically calculates the impact of mutations on a per-residue basis for all residues of a protein-protein interaction, achieving a precision similar to that of PARE. Our calculations show that electrostatics play a marginal role (<10 fold) in determining the rate of association for about half of the complexes in the database. Strong electrostatic steering, which results in an increase of over 100-fold in k(on), was calculated for about 25% of the complexes. Applying HyPare to all 68 complexes in the database shows that a small number of residues are hotspots for association. About 40% of the hotspots are calculated to increase the rate of association upon mutation, and thus increase binding affinity. This is a much higher ratio than found for hotspots for dissociation, where the large majority cause weaker binding. About 40% of the hotspots are located outside the physical boundary of the binding site, making them ideal candidates for protein engineering. Our data shows that a majority of protein-protein complexes are not optimized for fast association. Hotspots are not evenly distributed between all types of amino acids. About 75% of all hotspots are of charged residues. This is understandable, as a charge-reverse mutant changes the total charge by 2. The small number of hydrophobic residues that are hotspots upon mutation probably relates to their location and surrounding. For
  • Peleg-Shulman T., Roisman L., Zupkovitz G. & Schreiber G. 2004. Optimizing the binding affinity of a carrier protein - A case study on the interaction between soluble ifnar2 and interferon beta Journal of Biological Chemistry. 2004 Apr , 279 (17):18046-18053.

    [Abstract]

    Prolonging the circulatory half-life of low mass protein drugs can be achieved either by administration of a pro-drug or through co-injection with a carrier protein that will slowly release the active protein. The rate of release is concentration and affinity dependent. The optimal relationship between these two in prolonging the half-life of a pro-drug is the focus of this work. Interferon (IFN) beta is one of the most widely used protein drugs in the clinic. Here, we show that the circulatory half-life of IFNbeta can be significantly extended by coadministration with the extracellular domain of the IFN receptor ifnar2 (ifnar2-EC). To investigate the concentration/ affinity relation, a range of tighter binding ifnar2-EC mutants was designed that bind IFNbeta, but not IFNalpha2, up to 50-fold tighter compared with the wild-type ifnar2-EC. This increased affinity is related to a slower dissociation rate, whereas the association of IFNbeta with ifnar2-EC is already near optimum. Using the wild-type and mutant receptors, we investigated their potential in occluding IFNbeta from circulation in a tissue culture assay, as well as in rats. To determine the potential of ifnar2-EC as a carrier protein, we co-administered a mixture of IFNbeta and ifnar2-EC to rats both intravenously and subcutaneously, and followed the blood plasma concentrations of IFNbeta over time. The tighter binding ifnar2-EC mutant had a clear advantage in prolonging the half-life of IFNbeta in circulation, even when lower protein concentrations were administered. A numerical simulation of the in vivo data demonstrates that the optimal binding affinity of a carrier protein is around the concentration needed to obtain optimal activity of the ligand.
  • Kozer N. & Schreiber G. 2004. Effect of crowding on protein - Protein association rates: Fundamental differences between low and high mass crowding agents Journal of Molecular Biology. 2004 Feb , 336 (3):763-774.

    [Abstract]

    Physiological media constitutes a crowded environment that serves as the field of action for protein-protein interaction in vivo. Measuring protein-protein interaction in crowded solutions can mimic this environment. In this work we follow the process of protein-protein association and its rate constants (k(on)) of the beta-lactamase (TEM)-beta-lactamase inhibitor protein (BLIP) complex in crowded solution using both low and high molecular mass crowding agents. In all crowded solutions (0-40% (w/w) of ethylene glycol (EG), poly(ethylene glycol) (PEG) 200, 1000, 3350, 8000 Da Ficoll-70 and Haemaccel the measured absolute k(on), but not k(off) values, were found to be slower as compared to buffer. However, there is a fundamental difference between low and high mass crowding agents. In the presence of low mass crowding agents and Haemaccel k(on) depends inversely on the solution viscosity. In high mass polymer solutions k(on) changes only slightly, even at viscosities 12-fold higher than water. The border between low and high molecular mass polymers is sharp and is dictated by the ratio between the polymer length (L) and its persistence length (L-p). Polymers that are long enough to form a flexible coil (L/L-p > 2) behave as high molecular mass polymers and those who are unable to do so (L/L-p <2) behave as low molecular mass polymers. We concluded that although polymers solution are crowded, this property is not uniform; i.e. there are areas in the solution that contain bulk water, and in these areas proteins can diffuse and associate almost as if they were in diluted environment. This porous medium may be taken as mimicking some aspects of the cellular environment, where many of the macromolecules are organized along membranes and the cytoskeleton. To determine the contribution of electrostatic attraction between proteins in crowded milieu, we followed k(on) of wt-TEM and three BLIP analogs with up to 100-fold increased values of k(on) due to electrostatic steeri
  • Peleg-Shulman T., Tsubery H., Mironchik M., Fridkin M., Schreiber G. & Shechter Y. 2004. Reversible PEGylation: A novel technology to release native interferon alpha 2 over a prolonged time period Journal of Medicinal Chemistry. 2004 Sept , 47 (20):4897-4904.

    [Abstract]

    Many peptide and protein drugs have a short circulatory half-life in vivo. The covalent attachment of polyethylene glycol (PEG) chains (PEGylation) can overcome this deficiency, but pegylated peptides and proteins are often inactive. In this study, we present a novel PEG-IFNalpha2 conjugate, PEG(40)-FMS-IFNalpha2, capable of regenerating native interferon alpha2 (IFNalpha2) at a slow rate under physiological conditions. A 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) containing bifunctional reagent, MAL-FMS-NHS, has been synthesized, enabling the linkage of a 40 kDa PEG-SH to IFNalpha2 through a slowly hydrolyzable bond. By use of a BIAcore binding assay, the in vitro rate of regeneration of native interferon was estimated to have a half-life of 65 h. Following subcutaneous administration to rats and monitoring circulating antiviral activity, active IFNalpha2 levels peaked at 50 h, with substantial levels still being detected 200 h after administration. This value contrasts with a half-life of about I h measured for unmodified interferon. The concentration of active IFNalpha2 scaled linearly with the quantity injected. Comparing subcutaneous to intravenous administration of PEG(40)-FMS-IFNalpha2, we found that the long circulatory lifetime of IFNalpha2 was affected both by the slow rate of absorption of the PEGylated protein from the subcutaneous volume and by the slow rate of discharge from the PEG in circulation. A numerical simulation of the results was in good agreement with the results observed in vivo. The pharmacokinetic profile of this novel IFNalpha2 conjugate combines a prolonged maintenance in vivo with the regeneration of active-native IFNalpha2, ensuring ready access to peripheral tissues and thus an overall advantage over currently used formulations.
  • Gottschalk K., Neuvirth H. & Schreiber G. 2004. A novel method for scoring of docked protein complexes using predicted binding sites protein-protein binding sites PROTEIN ENGINEERING DESIGN & SELECTION. 2004 Feb , 17 (2):183-189.

    [Abstract]

    Docking algorithms produce many possible structures of a protein-protein complex. In most cases some of them resemble the correct structure within an r.m.s.d. of
  • Neuvirth H., Raz R. & Schreiber G. 2004. ProMate: A structure based prediction program to identify the location of protein-protein binding sites Journal of Molecular Biology. 2004 Apr , 338 (1):181-199.

    [Abstract]

    Is the whole protein surface available for interaction with other proteins, or are specific sites pre-assigned according to their biophysical and structural character? And if so, is it possible to predict the location of the binding site from the surface properties? These questions are answered quantitatively by probing the surfaces of proteins using spheres of radius of 10 Angstrom on a database (DB) of 57 unique, non-homologous proteins involved in heteromeric, transient protein-protein interactions for which the structures of both the unbound and bound states were determined. In structural terms, we found the binding site to have a preference for beta-sheets and for relatively long non-structured chains, but not for alpha-helices. Chemically, aromatic side-chains show a clear preference for binding sites. While the hydrophobic and polar content of the interface is similar to the rest of the surface, hydrophobic and polar residues tend to cluster in interfaces. In the crystal, the binding site has more bound water molecules surrounding it, and a lower B-factor already in the unbound protein. The same biophysical properties were found to hold for the unbound and bound DBs. All the significant interface properties were combined into ProMate, an interface prediction program. This was followed by an optimization step to choose the best combination of properties, as many of them are correlated. During optimization and prediction, the tested proteins were not used for data collection, to avoid over-fitting. The prediction algorithm is fully automated, and is used to predict the location of potential binding sites on unbound proteins with known structures. The algorithm is able to successfully predict the location of the interface for about 70% of the proteins. The success rate of the predictor was equal whether applied on the unbound DB or on the disjoint bound DB. A prediction is assumed correct if over half of the predicted continuous interface patch is indeed inte
  • Kiel C., Selzer T., Shaul Y., Schreiber G. & Herrmann C. 2004. Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex Proceedings of the National Academy of Sciences of the United States of America. 2004 Jun , 101 (25):9223-9228.

    [Abstract]

    Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by Phi analysis of the encounter complex and transition state of the RalGDS(M26K,D47K,ES4K)/Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.
  • Chill J., Quadt S., Levy R., Schreiber G. & Anglister J. 2003. The human type I interferon receptor: NMR structure reveals the molecular basis of ligand binding Structure. 2003 Jul , 11 (7):791-802.

    [Abstract]

    The potent antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by a single receptor comprising two subunits, IFNAR1 and IFNAR2. The structure of the IFNAR2 IFN binding ecto-domain (IFNAR2-EC), the first helical cytokine receptor structure determined in solution, reveals the molecular basis for IFN binding. The atypical perpendicular orientation of its two fibronectin domains explains the lack of C domain involvement in ligand binding. A model of the IFNAR2-EC/IFNalpha2 complex based on double mutant cycle-derived constraints uncovers an extensive and predominantly aliphatic hydrophobic patch on the receptor that interacts with a matching hydrophobic surface of IFNalpha2. An adjacent motif of alternating charged side chains guides the two proteins into a tight complex. The binding interface may account for crossreactivity and ligand specificity of the receptor. This molecular description of IFN binding should be invaluable for study and design of IFN-based biomedical agents.
  • Piehler J. & Schreiber G. 2003. Free Energy Landscapes in Protein-Protein Interactions Handbook of Cell Signaling. 2003 , 1-3 :27-31.

    [Abstract]

    [No abstract available]
  • Schreiber G. 2002. Kinetic studies of protein-protein interactions Current Opinion in Structural Biology. 2002 Feb , 12 (1):41-47.

    [Abstract]

    The structure of a protein-protein interaction, its affinity and thermodynamic characteristics depict a 'frozen' state of a complex. This picture ignores the kinetic nature of complex formation and dissociation, which are of major biological and biophysical interest. This review highlights recent advances in deciphering the kinetic pathway of protein-protein complexation, the nature of the encounter complex, transition state and intermediate along the reaction, and the effects of mutation, viscosity, pH and salt on association.
  • Chill J., Nivasch R., Levy R., Albeck S., Schreiber G. & Anglister J. 2002. The human interferon receptor: NMR-based modeling, mapping of the IFN-alpha 2 binding site, and observed ligand-induced tightening Biochemistry. 2002 Mar , 41 (11):3575-3585.

    [Abstract]

    The human interferon receptor (IFNAR) mediates the antiviral and antiproliferative activities of type I interferons (IFNs). This receptor is comprised of subunits IFNAR1 and IFNAR2, the latter exhibiting nanomolar affinity for IFNs. Here the extracellular domain of IFNAR2 (IFNAR2-EC), a soluble 25 kDa IFN-binding polypeptide, and its complex with IFN-alpha2 were studied using multidimensional NMR. IFNAR2-EC is comprised of two fibronectin-III (FN-III) domains connected by a helical hinge region. The deduced global fold was utilized to improve the alignment of IFNAR2-EC against structurally related receptors and to model its structure. A striking feature of IFNAR2-EC is the limited and localized deviations in chemical shifts exhibited upon ligand binding, observed for only 15% of its backbone H-1 and N-15 nuclei. Analysis of these deviations maps the IFN-alpha2 binding site upon IFNAR2-EC to a contiguous surface on the N-terminal domain, including the S3-S4 loop (residues 44-53), the S5-S6 loop and S6 beta-strand (residues 74-82), and the S7 beta-strand and the hinge region (residues 95-105). The C-terminal domain contributes only marginally to ligand binding, and no change in the hypothesized interdomain interface is observed. The proposed binding domain encompasses all residues implicated by mutagenesis studies in IFN binding, and suggests adjacent residues cooperate in forming the binding surface. D2O-exchange experiments indicate that binding of IFN-alpha2 induces tightening of the N-terminal domain of IFNAR2-EC. This increase in receptor rigidity may play an important role in initiating the intracellular stage of the IFN signaling cascade.
  • Piehler J. & Schreiber G. 2001. Fast transient cytokine-receptor interactions monitored in real time by reflectometric interference spectroscopy Analytical Biochemistry. 2001 Feb , 289 (2):173-186.

    [Abstract]

    Investigating protein-protein interactions by mutational analysis requires practical techniques for quantifying rate constants and equilibrium constants over several orders of magnitude with reasonably high sample throughput. We have employed spectroscopic interferometry for label-free monitoring of the interaction between the cytokine interferon alpha2 (IFN alpha2) and the extracellular domain of its receptor ifnar2 (ifnar2-EC), We implemented a versatile surface chemistry for the glass substrate of this transducer for covalent immobilization of proteins. Affinity capturing with a monoclonal anti-ifnar2-EC antibody (mAb) followed by crosslinking with a second, noncompetitive mAb provided stable, but still reversible, immobilization of ifnar2-EC, We measured kinetics and affinity of numerous of mutants of IFN alpha2 and ifnar2-EC. Dissociation rate constants up to 0.3 s(-1) and association rate constants up to 3 x 10(6) M(-)1 s(-1) were resolved by the system. Dissociation constants down to 200 muM were measured with protein concentrations up to 50 muM without no background signal or nonspecific binding. The instrument detection limit is similar to 10 pm without the need for temperature stabilization or referencing channels. The system proved effective for large-scale mutational analysis involving alanine scanning mutagenesis and double mutant cycles. (C) 2001 Academic Press.
  • Frisch C., Fersht A. & Schreiber G. 2001. Experimental assignment of the structure of the transition state for the association of barnase and barstar Journal of Molecular Biology. 2001 Apr , 308 (1):69-77.

    [Abstract]

    Association of a protein complex follows a two step reaction mechanism, with the first step being the formation of an encounter complex which evolves into the final complex. Here we present new experimental data for the association of the bacterial ribonuclease barnase and its polypeptide inhibitor barstar which shed light on the thermodynamics and structure of the transition state and preceding encounter complex of association at diminishing electrostatic attraction. We show that the activation entropy at the transition state is close to zero, with the activation enthalpy being equal to the free energy of binding. This observation was independent of the magnitude of the mutual electrostatic attraction, which were altered by mutagenesis or by addition of salt. The low activation entropy implies that the transition state is mostly solvated at all ionic strengths. The structure of the transition state was probed by measuring pairwise interaction energies using double-mutant-cycles. While at low ionic strength all proximal charge-pairs form contacts, at high salt only a subset of these interactions are maintained. More specifically, charge-charge interactions between partially buried residues are lost, while exposed charged residues maintain their ability to form specific interactions even at the highest salt concentration. Uncharged residues do not interact at any ionic strength. The results presented here suggest that the barnase-barstar binding sites are correctly aligned during the transition state even at diminishing electrostatic attraction, although specific short range interactions of uncharged residues are not yet formed. Furthermore, most of the interface desolvation (which contributes to the entropy of the system) has not yet occurred. This picture seems to be valid at low and high salt. However, at high salt, interactions of the activated complex are limited to a more restricted set of residues which are easier approached during diffusion, prior to final
  • Selzer T. & Schreiber G. 2001. New insights into the mechanism of protein-protein association Proteins-Structure Function And Genetics. 2001 Nov , 45 (3):190-198.

    [Abstract]

    Association of a protein complex follows a two-step mechanism, with the first step being the formation of an encounter complex that evolves into the final complex. Here, we analyze recent experimental data of the association of TEM1-beta -lactamase with BLIP using theoretical calculations and simulation. We show that the calculated Debye-Huckel energy of interaction for a pair of proteins during association resembles an energy funnel, with the final complex at the minima. All attraction is lost at inter-protein distances of 20 Angstrom, or rotation angles of > 60 degrees from the orientation of the final complex. For faster-associating protein complexes, the energy funnel deepens and its volume increases. Mutations with the largest impact on association (hotspots for association) have the largest effect on the size and depth of the energy funnel. Analyzing existing evidence, we suggest that the transition state along the association pathway is the formation of the final complex from the encounter complex. Consequently, pairs of proteins forming an encounter complex will tend to dissociate more readily than to evolve into the final complex. Increasing directional diffusion by increasing favorable electrostatic attraction results in a faster forming and slower dissociating encounter complex. The possible applicability of electrostatic calculations for protein-protein docking is discussed. (C) 2001 Wiley-Liss,Inc.
  • Roisman L., Piehler J., Trosset J., Scheraga H. & Schreiber G. 2001. Structure of the interferon-receptor complex determined by distance constraints from double-mutant cycles and flexible docking Proceedings of the National Academy of Sciences of the United States of America. 2001 Nov , 98 (23):13231-13236.

    [Abstract]

    The pleiotropic activity of type I interferons has been attributed to the specific interaction of IFN with the cell-surface receptor components ifnar1 and ifnar2. To date, the structure of IFIN has been solved, but not that of the receptor or the complex. In this study, the structure of the IFN-alpha2-ifnar2 complex was generated with a docking procedure, using nuclear Overhauser effect-like distance constraints obtained from double-mutant cycle experiments. The interaction free energy between 13 residues of the ligand and 11 of the receptor was measured by double-mutant cycles. Of the 100 pairwise interactions probed, five pairs of residues were found to interact. These five interactions were incorporated as distance constraints into the flexible docking program PRODOCK by using fixed and movable energy-gradient grids attached to the receptor and ligand, respectively. Multistart minimization and Monte Carlo minimization docking of IFN-alpha2 onto ifnar2 converged to a well-defined average structure, with the five distance constraints being satisfied. Furthermore, no structural artifacts or intraloop energy strain were observed. The mutual binding sites on IFN-alpha2 and ifnar2 predicted from the model showed an almost complete superposition with the ones determined from mutagenesis studies. Based on this structure, differences in IFN-alpha2 versus IFN-beta binding are discussed.
  • Shechter Y., Preciado-Patt L., Schreiber G. & Fridkin M. 2001. Prolonging the half-life of human interferon-alpha 2 in circulation: Design, preparation, and analysis of (2-sulfo-9-fluorenylmethoxycarbonyl)(7)-interferon-alpha 2 Proceedings of the National Academy of Sciences of the United States of America. 2001 Jan , 98 (3):1212-1217.

    [Abstract]

    Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-alpha2. The derivative thus obtained (FMS7-IFN-alpha2) has approximate to4% the biological potency and 33 +/- 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS7-IFN-alpha2 undergoes time-dependent spontaneous hydrolysis, generating active interferon with t(1/2) values of 24 +/- 2 h at pH 8.5 and 98 +/- 10 h at pH 7.4. When native IFN-alpha2 is intravenously administered to mice, circulating antiviral activity is maintained for a short duration and then declines with t(1/2) = 4 +/- 0.5 h, reaching undetectable values at approximate to 18 h after administration. With intravenously administered FMS7-IFN-alpha2, there is a lag period of 2 h, followed by a progressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t(1/2) = 35 +/- 4 h. FMS7-IFN-alpha2 is resistant to cu-chymotrypsin digest and to proteolytic inactivation by human serum proteases in vitro. We have thus introduced here an inactive IFN-alpha2 derivative, which is resistant to in site inactivation and has the capability of slowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes, FMS7-IFN-alpha2 maintains prolonged circulating antiviral activity in mice, exceeding 7-8 times the activity of intravenously administered native cytokine.
  • Albeck S., Unger R. & Schreiber G. 2000. Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges Journal of Molecular Biology. 2000 May , 298 (3):503-520.

    [Abstract]

    An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interactions between TEM-1-beta-lactamase and its protein inhibitor, BLIP. The selected interactions (two salt bridges and two hydrogen bonds) all involving BLIP-D49, define a distinct binding unit. The penta mutant, where all side-chains constructing the binding unit were mutated to Ala, was used-as a reference state to which combinations of sidechains were introduced. At first, pairs of interacting residues were added allowing the determination of interaction energies in the absence of neighbors, using double mutant cycles. Addition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neutral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol(-1) each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal mol(-1). Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors. These results demonstrate the importance of forming net-works of buried salt bridges. We present theoretical electrostatic calculations which predict the observed mode of cooperativity, and suggest that the cooperative networking effect results from the favorable contribution of the protein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the hive salt bridges, and to a lesser extent for the two hydrogen bonds, is shown. The data analysis was performed on values of Delta Delta
  • Selzer T., Albeck S. & Schreiber G. 2000. Rational design of faster associating and tighter binding protein complexes Nature Structural Biology. 2000 Jul , 7 (7):537-541.

    [Abstract]

    A protein design strategy was developed to specifically enhance the rate of association (k(on)) between a pair of proteins without affecting the rate of dissociation (K-off). The method is based on increasing the electrostatic attraction between the proteins by incorporating charged residues in the vicinity of the binding interface. The contribution of mutations towards the rate of association was calculated using a newly developed computer algorithm, which predicted accurately the rate of association of mutant protein complexes relative to the wild type. Using this design strategy, the rate of association and the affinity between TEM1 beta-lactamase and its protein inhibitor BLIP was enhanced 250-fold, while the dissociation rate constant was unchanged. The results emphasize that long range electrostatic forces specifically alter k(on), but do not effect k(off). The design strategy presented here is applicable for increasing rates of association and affinities of protein complexes in general.
  • Piehler J., Roisman L. & Schreiber G. 2000. New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface Journal of Biological Chemistry. 2000 Dec , 275 (51):40425-40433.

    [Abstract]

    Type I interferons bind to two cell surface receptors, ifnar1 and ifnar2, as the first step in the activation of several signal transduction pathways that elicit an antiviral state and an anti-proliferative response. Here, we quantitatively mapped the complete binding region of ifnar2 on interferon (IFN)alpha2 by 35 individual mutations to alanine and isosteric residues. Of the six "hot-spot" residues identified (Leu-30, Arg-33, Arg-144, Ala-145, Met-148, and Arg-149), four are located on the E-helix, which is located at the center of the binding site flanked by residues on the A-helix and the AD-loop. The contribution of residues of the D-helix, which have been previously implicated in binding, proved to be marginal for the interaction with the extracellular domain of ifnar2. Interestingly, the ifnar2 binding site overlaps the largest continuous hydrophobic patch on IFN alpha2. Thus, hydrophobic interactions seem to play a significant role stabilizing this interaction, with the charged residues contributing toward the rapid association of the complex. Relating the anti-viral and anti-proliferative activity of the various interferon mutants with their affinity toward ifnar2 results in linear function over the whole range of affinities investigated, suggesting that ifnar2 binding is the rate-determining step in cellular activation. Dose-time analysis of the anti-viral response revealed that shortening the incubation time of low-level activation cannot be compensated by higher IFN doses. Considering the strict dependence of the cellular response on affinity, these results suggest that for maintaining transcription of IFN-responsive genes over a longer time period, low but continuous signaling through the IFN receptor is essential.
  • Piehler J. & Schreiber G. 1999. Biophysical analysis of the interaction of human ifnar2 expressed in E-coli with IFN alpha 2 Journal of Molecular Biology. 1999 May , 289 (1):57-67.

    [Abstract]

    Type I interferons are cytokines which activate an anti-viral response by binding to two specific cell surface receptors, ifnar1 and ifnar2. Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha 2 (IFN alpha 2). The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibits antiviral activity of IFN alpha 2. The stoichiometry of binding IFN alpha 2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction is 10 nM, which is similar to the affinity measured for the cell surface-bound ifnar2 receptor. No difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectroscopy. However, the binding kinetics as measured in homogeneous phase by fluorescence dequenching was about three times faster than that measured on a sensor surface. The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggest a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pK(a) of 6.7. The surface properties of the IFN alpha 2 binding surface on ifnar2 were interpreted according to the pH and salt dependence of the interaction. (C) 1999 Academic Press.
  • Albeck S. & Schreiber G. 1999. Biophysical characterization of the interaction of the beta-lactamase TEM-1 with its protein inhibitor BLIP Biochemistry. 1999 Jan , 38 (1):11-21.

    [Abstract]

    BLIP is a secreted protein from Streptomyces clavuligerus that inhibits a wide range of beta-lactamases. Here we investigate the tight interaction of BLIP, expressed heterologousely in E. coli, with TEM-1. Kinetic and thermodynamic constants were determined using methods with the proteins either in a homogeneous or in a heterogeneous phase. While values of Delta Delta G((mut-wt)) are similar whether measured by fluorescence quench, enzyme inhibition, or surface plasmon resonance, absolute values of Delta G and kinetic constants vary. Association and dissociation rate constants of 10(5) M-1 s(-1) and 10(-4) s(-1) respectively, and a nanomolar affinity were determined for the wild-type proteins. The highest affinity is measured at pH 7.5, with a decreasing association rate constant at higher pH values, and an increasing dissociation rate constant at lower pH values. The marginal effect of salt on the kinetics of binding, as well as the calculated surface potentials, suggests a limited role for electrostatic forces in guiding this reaction. Still, mutations of interfacial residues affect the rate of association significantly, so that an increase in the net negative charge on either protein reduces the association rate constant. We show that simple electrostatic rules can explain this behavior. BLIP inhibits the catalytic activity of TEM-1 by binding its active site. Yet, mutations of active site residues on TEM-1 only have a moderate though cooperative effect on the binding energy. This can be explained in light of the peripheral location of the active site in the interface between the two proteins.
  • Piehler J. & Schreiber G. 1999. Mutational and structural analysis of the binding interface between type I interferons and their receptor ifnar2 Journal of Molecular Biology. 1999 Nov , 294 (1):223-237.

    [Abstract]

    Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFN alpha 2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2 residues T46, I47 and M48 were identified as hot-spots in the interaction with IFN alpha 2. For another ten residues on ifnar2, significant contribution of interaction Energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFN alpha 2. Although IFN alpha 2 and IFN beta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling. (C) 1999 Academic Press.
  • Selzer T. & Schreiber G. 1999. Predicting the rate enhancement of protein complex formation from the electrostatic energy of interaction Journal of Molecular Biology. 1999 Mar , 287 (2):409-419.

    [Abstract]

    The rate of association of proteins is dictated by diffusion, but can be enhanced by favorable electrostatic forces. Here the relationship between the electrostatic energy of interaction, and the kinetics of protein-complex formation was analyzed for the protein pairs of: hirudin-thrombin, acetylcholinesterase-fasciculin and barnase-barstar, and for a panel of point mutants of these proteins. Electrostatic energies of interaction were calculated as the difference between the electrostatic energy of the complex and the sum of the energies of the two individual proteins, using the computer simulation package DelPhi. Calculated electrostatic energies of interaction were compared to experimentally determined rates of association. One kcal/mol of Coulombic interaction energy increased the rate of association by a factor of 2.8, independent of the protein-complex or mutant analyzed. Electrostatic energies of interaction were also determined from the salt dependence of the association rate constant, using the same basic equation as for the theoretical calculation. A Bronsted analysis of the electrostatic energies of interactions plotted versus experimentally determined In(rate)s of association shows a linear relation between the two, with a beta value close to 1. This is interpreted as the energy of the transition state varies according to the electrostatic interaction energy, fitting a two state model for the association reaction. Calculating electrostatic rate enhancement from the electrostatic interaction energy can be used as a powerful tool to design protein complexes with altered rates of association and affinities. (C) 1999 Academic Press.
  • Vijayakumar M., Wong K., Schreiber G., Fersht A., Szabo A. & Zhou H. 1998. Electrostatic enhancement of diffusion-controlled protein-protein association: Comparison of theory and experiment on barnase and barstar Journal of Molecular Biology. 1998 May , 278 (5):1015-1024.

    [Abstract]

    The electrostatic enhancement of the association rate of barnase and barstar is calculated using a transition-state theory like expression and atomic-detail modeling of the protein molecules. This expression predicts that the rate enhancement is simply the average Boltzmann factor in the region of configurational space where association occurs instantaneously in the diffusion-controlled limit. Based on experimental evidence, this "transition state" is defined by configurations in which, relative to the stereospecifically bound complex, the two proteins are shifted apart by similar to 8 Angstrom (so a layer of water can be accommodated in the interface) and the two binding surfaces are rotated away by 0 degrees to 3 degrees. The values of the average Boltzmann factor, calculated by solving the Poisson-Boltzmann equation, for the wild-type complex and 16 complexes with single mutations are found to correlate well with experimental results for the electrostatic rate enhancement. The predicted rate enhancement is found to be somewhat insensitive to the precise definition of the transition state, due to the long-range nature of electrostatic interactions. The experimental ionic strength dependence of the rate enhancement is also reasonably reproduced. (C) 1998 Academic Press Limited.
  • Schreiber G., Frisch C. & Fersht A. 1997. The role of Glu73 of barnase in catalysis and the binding of barstar Journal of Molecular Biology. 1997 Jul , 270 (1):111-122.

    [Abstract]

    Barnase, a small extracellular ribonuclease from Bacillus amyloliquefaciens and its intracellular inhibitor barstar have co-evolved to bind tightly and rapidly. Barnase has also evolved to be catalytically active. The active site of barnase and its binding site for barstar use the same subset of amino acids. The exception is Glu73 (the general base in catalysis), which although located at the centre of the binding site, is separated by three ordered water molecules from barstar. We examined in this work the contribution of Glu73 to both catalysis and barstar binding. Truncation mutants of the general base (Glu73 --> Ala or Ser) retain a residual RNase activity of about 0.3% while mutants with larger hydrophobic replacements (Glu 73 --> Trp or Phe) have virtually no catalytic activity. This, and binding data of 3'-GMP with the different barnase mutants suggest that the loss in activity results from the elimination of the general base, which can be substituted to some extent by water or other polar sidechains in truncation mutants. All of the Glu73 mutations lead to a weakening of the free energy of complex formation with barstar by 1.4 to 3.0 kcal/mol (including Gln). This is surprising, since Glu73 does not interact directly with barstar and there is an electrostatic repulsion between Glu73 on barnase and the negatively charged binding surface of barstar. A newly developed method of constructing double mutant cycles between multiple mutations at the same site appears to pinpoint a favourable interaction between Glu73 and one of its nearest neighbours in barstar, Asp39. The coupling energy between those residues is presumably indirect: the carboxylate of Glu73 organizes neighbouring positively charged groups in barnase, Lys27, Arg83, and Arg87 to interact with Asp39 in barstar. This emphasizes that an apparent interaction between a pair of residues as measured with double mutant cycles is the sum of their direct and indirect interactions. (C) 1997 Academic Press L
  • Schreiber G. & Fersht A. 1996. Rapid, electrostatically assisted association of proteins Nature Structural Biology. 1996 May , 3 (5):427-431.

    [Abstract]

    The rapid association of barnase and its intracellular inhibitor bar star has been analysed from the effects of mutagenesis and electrostatic screening. A basal association rate constant of 10(5) M(-1) s(-1) is increased to over 5 x 10(9) M(-1) s(-1) by electrostatic forces. The association between the oppositely charged proteins proceeds through the rate-determining formation of an early, weakly specific complex, which is dominated by long-range electrostatic interactions, followed by precise docking to form the high affinity complex. This mode of binding is likely to be used widely in nature to increase association rate constants between molecules and its principles may be used for protein design.
  • Frisch, C., G. Schreiber, and A.R. Fersht 1995. Characterization of in vitro oxidized barstar FEBS Lett, 1995. 370(3): p. 273-7. 1995 , .
  • Martinez, J.C., V.V. Filimonov, P.L. Mateo, G. Schreiber, and A.R. Fersht 1995. A calorimetric study of the thermal stability of barstar and its interaction with barnase Biochemistry. 1995 , 34 (15):5224-33.
  • Schreiber, G. and A.R. Fersht 1995. Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles J Mol Biol. 1995 , 248 (2):478-86.
  • SCHREIBER G, RON EZ, GLASER G 1995. PPGPP-MEDIATED REGULATION OF DNA-REPLICATION AND CELL-DIVISION IN ESCHERICHIA-COLI. CURRENT MICROBIOLOGY. 1995 , 30 (10):27-32.
  • Buckle, A.M., G. Schreiber, and A.R. Fersht 1994. Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution Biochemistry. 1994 , 33 (30):8878-89.
  • AVIV M, GILADI H, SCHREIBER G, OPPENHEIM AB, GLASER G 1994. EXPRESSION OF THE GENES-CODING FOR THE ESCHERICHIA-COLI INTEGRATION HOST FACTOR ARE CONTROLLED BY GROWTH-PHASE, RPOS, PPGPP AND BY AUTOREGULATION MOLECULAR MICROBIOLOGY. 1994 , 14 (30):1021-1031.
  • Schreiber, G., A.M. Buckle, and A.R. Fersht 1994. Stability and function: two constraints in the evolution of barstar and other proteins Structure. 1994 , 2 (10):945-51..
  • Schreiber, G. and A.R. Fersht 1993. Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering Biochemistry. 1993 , 32 (19):5145-50..
  • SCHREIBER G, METZGER S, AIZENMAN E, ROZA S, CASHEL M, GLASER G, 1993. OVEREXPRESSION OF THE RELA GENE IN ESCHERICHIA-COLI. Biochemistry. 1993 , 32 (41):11195-203..
  • Schreiber, G. and A.R. Fersht 1991. The refolding of cis- and trans-peptidylprolyl isomers of barstar JBC. 1991 , 266 :3760-3767.
  • METZGER S, SCHREIBER G, AIZENMAN E, CASHEL M, GLASER G, 1989. CHARACTERIZATION OF THE RELA1 MUTATION AND A COMPARISON OF RELA1 WITH NEW RELA NULL ALLELES IN ESCHERICHIA-COLI. JBC. 1989 , 264 :21146-21152..
  • METZGER S, BENDROR I, AIZENMAN E, SCHREIBER G, TOONE M, FRIESEN JD, CASHEL M, GLASER G, 1988. THE NUCLEOTIDE-SEQUENCE AND CHARACTERIZATION OF THE RELA GENE OF ESCHERICHIA-COLI. JBC. 1988 , 263 :15699-15704..