Abstract
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.
Highlights
Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character
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
Using the projection approximation method [25, 26], we estimated that the docked and undocked states of DnaK would have a difference in CCSs large enough to be separated by ion-mobility native mass spectrometry (IMMS). (Note that because the constructs of DnaK analyzed by crystallography and NMR lack the C-terminal domain and part of the ␣substrate-binding domain (SBD) (Fig. 1A), the projection approximation was applied only to evaluate the differences on CCS between the docked and undocked states.) the calculated CCSs were significantly different: 4193 Å2 for the ATP-bound (from PDB entry 4B9Q [8]) and 4681 Å2 for the ADP/substrate-bound (from PDB entry 2KHO [6])
Summary
Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. In addition to the interdomain rearrangements that occur upon nucleotide binding and hydrolysis, the helical lid is dynamic and confers functional plasticity to Hsp70 This plasticity allows Hsp70s to bind a variety of substrate conformations, from extended short peptides to near-native protein intermediate states [12, 14, 15]. The present study deploys two complementary techniques, ion-mobility native mass spectrometry (IMMS) and double electron-electron resonance (DEER), to answer these questions and elucidate the conformational ensemble of DnaK under biologically relevant ligand-bound conditions (ATP, ADP/substrate, and ATP/substrate). IMMS and DEER have allowed us to survey the arrangement of the conformational ensembles of DnaK under different ligand-bound conditions and to identify sparsely populated conformers These approaches establish that ATP/ substrate-bound DnaK populates a discrete partially docked state
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