Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance

Journal article

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.

Authors: Lai, A; Clerico, E; Blackburn, M; Patel, N; Robinson, C

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: American Society for Biochemistry and Molecular Biology On
• Journal: Journal of Biological Chemistry
• Volume: 292
• Issue: 21
• Pages: 8773-8785
• Publication date: 2017-04-01
• Acceptance date: 2017-04-12
• DOI: 10.1074/jbc.M116.770404
• EISSN: 1083-351X
• ISSN: 0021-9258
• Language: English
• Keywords: Hsp70; energy landscape; molecular chaperone; allosteric regulation; double electron-electron resonance; ion-mobility mass spectrometry; chaperone DnaK (DnaK); conformational change; 70-kilodalton heat shock protein (Hsp70)