Hsp70 chaperones occur in all organisms and essentially all cellular compartments. Among their wide array of essential cellular functions, they facilitate folding of newly synthesized proteins;protect cells from damage such as aggregation that can occur under stress conditions;help to target proteins to extra- cytoplasmic locations;and facilitate assembly and disassembly of macromolecular complexes. All of these functions rely on the ability of Hsp70s to bind unfolded regions of a protein substrate, and to release their substrates upon allosteric binding of ATP. The research proposed focuses on the fundamental molecular mechanism of Hsp70 allostery. The work proposed builds on exciting recent results: We showed in the last project period that both the ATPase domain and the substrate-binding domain (SBD) of the paradigmatic E. coli Hsp70 DnaK undergo major conformational changes upon ATP binding, and we gained understanding of the allosteric remodeling of these domains. Our results led us to a model for interdomain allosteric communication in DnaK that has been validated by a recent structure from the Hendrickson lab of a related chaperone, Sse1, the yeast Hsp110 [Q. Liu and W. A. Hendrickson, Cell 131, 106-1202007)].
Our specific aims are: to refine the current Sse1-based homology model of ATP-bound DnaK and to use this model, together with our knowledge about the ADP-bound state of DnaK, to elucidate the mechanism of allosteric interdomain communication in this Hsp70 molecular chaperone;to assess the generality of results on DnaK and develop general principles about Hsp70 allosteric function;to explore how the allosteric conformational changes in DnaK are modulated by interaction with co-chaperones DnaJ and GrpE. We will utilize new NMR strategies applicable to large molecules in order to analyze both structural and dynamic aspects of the allosteric conformational transitions in Hsp70s upon binding to their ligands and co-chaperones. Complementary data will be provided by time-resolved fluorescence energy transfer and electron spin resonance methods, as well as computational approaches based on sequence analysis, normal mode calculations, and ensemble-based thermodynamic dissection of ligand-mediated energetics. Hsp70s constitute relatively simple allosteric machines. Studying in detail their allosteric interdomain communication will shed light on the broader puzzle of how proteins harness ligand-binding energy to modulate binding and catalytic functions at a distance.

Public Health Relevance

Hsp70 molecular chaperones play key cellular roles under normal physiological conditions and enable cells to withstand stress such as heat shock. Hsp70s are known to be anti-apoptotic and up- regulated in tumors;ironically, their up-regulation is protective against neurodegenerative diseases caused by protein misfolding. The intimate involvement of Hsp70s in both normal and disease states has led to their emergence as possible therapeutic targets, but using heat shock proteins in a therapeutic capacity requires that we fully understand their mechanism of action, including how nucleotide modulates substrate binding and how interactions with co-chaperones modulate Hsp70 allostery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027616-33
Application #
8099473
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Chin, Jean
Project Start
1988-01-01
Project End
2012-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
33
Fiscal Year
2011
Total Cost
$273,372
Indirect Cost
Name
University of Massachusetts Amherst
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
English, Charles A; Sherman, Woody; Meng, Wenli et al. (2017) The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains. J Biol Chem 292:14765-14774
Lai, Alex L; Clerico, Eugenia M; Blackburn, Mandy E et al. (2017) Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance. J Biol Chem 292:8773-8785
Medus, Máximo Lopez; Gomez, Gabriela E; Zacchi, Lucía F et al. (2017) N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins. Sci Rep 7:8788
Hebert, Daniel N; Clerico, Eugenia M; Gierasch, Lila M (2016) Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences. Mol Cell 63:721-3
Clerico, Eugenia M; Tilitsky, Joseph M; Meng, Wenli et al. (2015) How hsp70 molecular machines interact with their substrates to mediate diverse physiological functions. J Mol Biol 427:1575-88
Zhuravleva, Anastasia; Gierasch, Lila M (2015) Substrate-binding domain conformational dynamics mediate Hsp70 allostery. Proc Natl Acad Sci U S A 112:E2865-73
Hong, Jiang; Gierasch, Lila M; Liu, Zhicheng (2015) Its preferential interactions with biopolymers account for diverse observed effects of trehalose. Biophys J 109:144-53
Theillet, Francois-Xavier; Binolfi, Andres; Frembgen-Kesner, Tamara et al. (2014) Physicochemical properties of cells and their effects on intrinsically disordered proteins (IDPs). Chem Rev 114:6661-714
Chien, Peter; Gierasch, Lila M (2014) Challenges and dreams: physics of weak interactions essential to life. Mol Biol Cell 25:3474-7
Gershenson, Anne; Gierasch, Lila M; Pastore, Annalisa et al. (2014) Energy landscapes of functional proteins are inherently risky. Nat Chem Biol 10:884-91

Showing the most recent 10 out of 83 publications