Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub(PT)), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub(PT), and we expect that its future uses will help define and investigate the ubiquitin interactome.
Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles.
Rosenzweig R, Sekhar A, Nagesh J, Kay LE.
Elife. 2017 Jul 14;6. pii: e28030. doi: 10.7554/eLife.28030.
The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state.
The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a H-1-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.
Sekhar A, Rosenzweig R, Bouvignies G, Kay L.E. (2016). Hsp70 Biases the Folding Pathways of Client Proteins. Proc Natl Acad Sci USA. 113:(20) 2794-801.
Rosenzweig R, Kay L.E. (2016). Solution NMR Spectroscopy Provides an Avenue For the Study of Functionally Dynamic Molecular Machines: the Example of Protein Disaggregation. J. Am. Chem. Soc. 138:(5) 1466-77.
Sekhar A, Rosenzweig R, Bouvignies G, Kay L.E. (2015). Mapping the Conformation of a Client Protein Through the Hsp70 Functional Cycle. Proc Natl Acad Sci USA. 112:(33) 10395-400.
Rosenzweig R, Farber P, Velyvis A, Rennella E, Latham M.P, Kay L.E. (2015). ClpB N-Terminal Domain Plays a Regulatory Role in Protein Disaggregation. Proc Natl Acad Sci USA. 112:(50) 6872-81.
Rosenzweig R, Moradi S, Zarrine-Afsar A, Glover J.R, Kay L.E. (2013). Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction. Science 339(6123): 1080-3
Rosenzweig R, Bronner V, Fushman D, Glickman M.H. (2012). Rpn1 and Rpn2 coordinate ubiquitin processing factors at the proteasome. J Biol Chem. 287 (18): 14659-71.
Deriziotis P, André R, Smith D.M, Goold R, Kinghorn K.J, Kristiansen M, Nathan J.A, Rosenzweig R, Krutauz D, Glickman M.H, Collinge J, Goldberg A.L, Tabrizi S.J. (2011). Misfolded PrP Impairs the UPS by Interaction With the 20S Proteasome and Inhibition of Substrate Entry. EMBO J. 30:(15) 3065-77.
Religa T.L, Ruschak A.M, Rosenzweig R, Kay L.E. (2011). Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supramolecular Protein Systems: Applications to the Proteasome and to the ClpP Protease. J. Am Chem Soc. 133:(23) 9063-8.
Rosenzweig R, Osmulski P.A, Gaczynska M, Glickman M.H. (2008). The Central Unit Within the 19S Regulatory Particle of the Proteasome. Nat Struct Mol Biol. 15:(6) 573-80.
Rosenzweig R, Glickman MH. (2008). Forging a proteasome alpha-ring with dedicated proteasome chaperones. Nat Struct Mol Biol. 5(3):218-20