Abstract
J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface—thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition—via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage.
Highlights
By transiently binding many different polypeptide substrates, Hsp70 chaperones assist in diverse cellular processes, from de novo protein folding to protein trafficking to disassembly of protein complexes
To obtain a structural model of the J-domain proteins (JDPs)-Hsp70 complex formed by Hsc20 and Ssq1, we combined protein-protein docking with all-atom molecular dynamics (MD) simulations
The J-domains bind at the nucleotide binding domain (NBD)/SBDβ,linker interface such that helix II predominantly interacts with the β-sheet region of NBD subdomain IIa and helix III with SBDβ (Fig 1)
Summary
By transiently binding many different polypeptide substrates, Hsp chaperones assist in diverse cellular processes, from de novo protein folding to protein trafficking to disassembly of protein complexes. They transiently interact with ATP-bound Hsp via their defining J-domain This interaction, in coordination with substrate binding, facilitates stimulation of Hsp70’s ATPase activity, which in turn drives the large scale conformational changes that stabilize substrate interaction [1,2,3,4]. The structure of both J-domains and Hsp70s are conserved. The helix II/III connecting loop includes a conserved histidine, proline, aspartate tripeptide (HPD) These invariant residues are critical for J-domain stimulation of Hsp70’s ATPase activity. Upon ATP hydrolysis, the domains disengage, and the substrate is “trapped” as the α subdomain of the SBD (SBDα) closes over the SBDβ substrate binding site [7,8,9]
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