Biophysical and kinetic studies of protein-protein interactions in vitro and in vivo »

Protein complexation is a kinetic process, where the formation of an encounter (transient) complex precedes the formation of the final complex. We studied the nature of the encounter complex and transition state for binding both in buffer and in a crowded environment, and developed in silico tools to design faster and tighter binding protein complexes. We provided experimental evidence on how electrostatic forces increase the rate of association through the stabilization of the encounter complex without affecting the final docking rate. Combining experiments and computer simulations, we showed that only some of the encounters are fruitful while other are futile and that the transition state for binding can be specific or diffusive. We designed mutations that enhanced the fruitful and specific encounters, and by this increased their rate of association. Surprisingly, in a crowded environment the association and dissociation rate constants are almost as rapid as in water, apparently due to the occluded volume effect. Studying binding kinetics in real-time in living cells showed that binding is as fast in cells as in vitro and that electrostatic forces have similar effects also in cells. This validates many years of in vitro studies on this subject. 

  1. Selzer T, Albeck S, Schreiber G. Rational design of faster associating and tighter binding protein complexes. Nat Struct Biol. 2000;7:537–541.
  2. Schreiber G, Haran G, Zhou HX. Fundamental Aspects of Protein-Protein Association Kinetics. Chem Rev. 2009;109:839–860.
  3. Phillip Y, Kiss V, Schreiber G. Protein-binding dynamics imaged in a living cell. Proc Natl Acad Sci U S A. 2012;109:1461–1466.

The molecular architecture of protein-protein interfaces »

A major interest in my laboratory is to decipher the physico-chemical nature of protein binding sites. Drawing the interface as a connected graph (network), with the amino-acids being the nodes, and the bonds being the edges showed that protein-protein interfaces are made of an aggregate of independent modules, with each module comprising a number of cooperatively interacting residues. The space between modules is occupied by interface water molecules, which we showed to be neutral in their contribution to binding. Experimental studies using a large number of mutants, as well as X-ray crystallography confirmed this interface architecture. Based on the modularity of the interface, we successfully re-designed the interface between a pair of proteins to high affinity and specificity in relation to the wild-type. 

  1. Reichmann D, Rahat O, Albeck S, Meged R, Dym O, Schreiber G. The modular architecture of protein-protein binding interfaces. Proc Natl Acad Sci U S A. 2005;102:57–62.
  2. Potapov V, Cohen M, Inbar Y, Schreiber G. Protein structure modelling and evaluation based on a 4-distance description of side-chain interactions. BMC Bioinformatics. 2010;11:374.
  3. Schreiber G, Keating AE. Protein binding specificity versus promiscuity. Curr Opin Struct Biol. 2011;21:50–61.

Development of bioinformatics tools »

The development of bioinformatics and computational tools are complementary to our wet lab work. We developed a popular predictor to locate the binding site between proteins, made a number of web based tools for better understanding of the architecture of proteins and interfaces, and used the gained knowledge to construct a new energy-function for protein modeling and design. All of our bioinformatics tools have a WEB based interface and are simple to use.

  1. Neuvirth H, Raz R, Schreiber G. ProMate: A Structure Based Prediction Program to Identify the Location of Protein-Protein Binding Sites. J Mol Biol. 2004;338:181–199.
  2. Reichmann D, Phillip Y, Carmi A, Schreiber G. On the contribution of water-mediated interactions to protein-complex stability. Biochemistry. 2008;47:1051–1060.
  3. Rahat O, Alon U, Levy Y, Schreiber G. Understanding hydrogen-bond patterns in proteins using network motifs. Bioinformatics. 2009;25:2921–2928.
  4. Potapov V, Cohen M, Inbar Y, Schreiber G. Protein structure modelling and evaluation based on a 4-distance description of side-chain interactions. BMC Bioinformatics. 2010;11:374.


Investigating the differential activities of type I interferons »

How can binding of similar ligands to the same receptor complex result in a variety of biological activities (antiviral, antiproliferative, immunomodulatory, etc)? To study the complexity of interferon signaling we applied the following research directions:

  1. Structure/function studies on the ligand receptor complex, including the determination of high-resolution structures of the individual components and of the complex bound to interferons;
  2. Studying a large set of interferon mutants that mimic the binding characteristics of the 17 members of the type I interferons, including the production of super agonist and antagonist interferons;
  3. Investigating the cross talk between binding affinity and receptor density, and how these affect activity;
  4. Using gene-arrays and high-throughput gene-knockdown to investigate how differences in receptor binding result in differetial signaling.

Our studies showed that differential activation relates to the stability of the ternary ligand-receptor complex, the time of activation, the concentration and affinity of the ligand and the density of the surface receptors. We show that the antiviral function is robust, rapidly initiated and maintained over a prolonged time, while, antiproliferative activity, which is a combination of apoptosis and cell cycle arrest, is tunable, and requires a prolonged time of activation. Our studies also resulted in the development of interferon agonists and antagonists. We developed the most potent interferon available, and showed its activity both in vitro, in vivo and in animal disease models. In addition, we developed an interferon antagonist, which can block specifically the interferon induced antiproliferative activity.

  1. Thomas C, Moraga I, Levin D, Krutzik PO, Podoplelova Y, Trejo A, Lee C, Yarden G, Vleck SE, Glenn JS et al. Structural Linkage between Ligand Discrimination and Receptor Activation by Type I Interferons. Cell. 2011;146:621–632.
  2. Levin D, Harari D, Schreiber G. Stochastic Receptor Expression Determines Cell Fate upon Interferon Treatment. Mol Cell Biol. 2011;31:3252–3266.
  3. Piehler J, Thomas C, Christopher Garcia K, Schreiber G. Structural and dynamic determinants of type I interferon receptor assembly and their functional interpretation. Immunol Rev. 2012;250:317–334.
  4. Apelbaum A, Yarden G, Warszawski S, Harari D, Schreiber G. Type I Interferons Induce Apoptosis by Balancing cFLIP and Caspase-8 Independent of Death Ligands. Mol Cell Biol. 2013;33:800–814.


Development of transgenic mice models to investigate human interferon action »

A major problem in studying human type I interferons is their low activity in mice. To overcome this problem we developed a transgenic mouse, which harbors also the receptors of human type I interferons. After validation, we investigated the effect of different type I interferons on specific disease models, particularly EAE. We found our agonist, which was altered to have a long half-life in the serum to be highly effective. We are currently using this model to further investigate the mechanism of action of interferons in EAE and testing it also on other disease models.