Two-step mechanism of J-domain action in driving Hsp70 function

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.

The Hsp70-Chaperone Machines in Bacteria

“…The J-domain binds on top of the interdomain linker that is important for the stimulation of the ATPase activity and interacts with NBD and SBDβ ( Vogel et al, 2006b ; Swain et al, 2007 ). It is positioned by electrostatic interaction between positively charged residues in the J-domain (R22, K26, R27, K48, K51) and negatively charged residues in the NBD (E206, D211, E217) and SBDβ (D477) as had been proposed based on NMR and computational data ( Ahmad et al, 2011 ; Malinverni et al, 2017 ; Tomiczek et al, 2020 ). Genetic screens had identified the highly conserved histidine-proline-aspartate (HPD) motif as essential for the functional interaction of the J-domain with Hsp70.…”

Interaction of client—the scaffold on which FeS clusters are build—with J-domain protein Hsc20 and its evolving Hsp70 partners

“…And, more fundamentally, how is any network fine-tuned to allow many different JDPs to effectively function with a single Hsp70? (Tomiczek et al, 2020). As in all JDP-Hsp70 interactions, the J-domain binds at the interface of NBD, β-SBD and interdomain linker formed when Ssq1 is in the ATP-bound conformation.…”

“…However, while the HPD is invariant, the residues of helix 2 and 3 forming the Hsp70 binding face are not conserved. Critical interfacial positions are occupied by very different amino acids, even in relatively closely related species ( Figures 5B,C ) ( Delewski et al, 2016 ; Tomiczek et al, 2020 ). Perhaps most importantly, these interfaces are networks of interconnecting residues—key residues on one side of an interface form contacts with more than one residue on the other side ( Figure 5B ).…”

“…FIGURE 5J-domain-Hsp70 interaction involves evolutionary variable interface. (A) Structural model of the S. cerevisiae Hsc20-Ssq1 (ATP) complex(Tomiczek et al, 2020). As in all JDP-Hsp70 interactions, the J-domain binds at the interface of NBD, β-SBD and interdomain linker formed when Ssq1 is in the ATP-bound conformation.…”

Multivalent protein–protein interactions are pivotal regulators of eukaryotic Hsp70 complexes

“…6 ) (Ungewickell et al 1997 ; Greene et al 1998 ; Matthias P. Mayer et al 1999 ; Suh et al 1999 ; Ahmad et al 2011 ; J. Jiang et al 2007 ; Kityk et al 2018 ). Mutations in these sites hinder association with Hsp70 and block the stimulation of ATP turnover (Kityk et al 2018 ; Tomiczek et al 2020 ), supporting their functional importance. The affinity of the J-domain for Hsp70s has been measured using a variety of approaches and is variably estimated to be between 0.07 and 0.54 μ M (Suh et al 1998 ; Suh et al 1999 ) or 5 to 10 μ M (Greene et al 1998 ).…”