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. are able to discriminate amongst their multiple JDP partners, thus enabling evolution of complex JDP/Hsp70 interaction networks. ResultsStructural model of the Hsc20-Ssq1 complex. To obtain a structural model of the JDP-Hsp70 complex formed by Hsc20 and Ssq1, we combined protein-protein docking with all-atom molecular dynamics (MD) simulations ( Supplementary Fig. S1). This approach revealed a dominant bound state that accounted for 56% of the bound population ( Fig. 1, Supplementary Fig. S2a). No other bound state accounted for more than 6% of the ensemble; these minor states rapidly interconverted and had a clear tendency to converge to the most populated bound state ( Supplementary Fig. S2b). The binding mode in this dominant state closely resembles the recently published X-ray structure of DnaJ's J-domain in complex with DnaK (DnaJ JD -DnaK) 22 . Helices II and III are oriented very similarly in the two complexes -with a backbone root mean square deviation (RMSD) of 4.17 Å ( Supplementary Fig. S3). More specifically, the J-domains bind at the NBD/SBDb,linker interface such that helix II predominantly interacts with the b-sheet region of NBD subdomain IIa and helix III with SBDb (Fig. 1). The HPD residues are positioned nearly identically in the two complexes, towards R167/R207 of NBD subdomain Ia in DnaK/Ssq1 (referred to as R NBD throughout), the arginine that previous analyses of DnaK established as important for allosteric transitions 14,20,21 . D HPD interferes in Hsp70 interdomain interactions by perturbing critical R NBD contacts.To address the question of the mechanism of J-domain action, we employed MD simulations that, unlike most other methods, allow studying of transient r...

Chaperoning shape-shifting tau in disease

“…This highly conserved domain is critical for JDP to function in tandem with Hsp70, with structurally disruptive mutations shown to impair Hsp70 binding and, by extension, inhibit Hsp70-based activity [61,62]. However, there is currently no direct evidence to suggest that the JD itself is involved in substrate recognition or binding, but rather appears to be coevolving with Hsp70 surfaces [63]. Importantly, the least understood function of JDP members is how these chaperones recognize their substrates.…”

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins

“…Genetic, biochemical, and structural data accumulated over the years provide a relatively comprehensive map of the J-domain-Hsp70 interface (23,24,(45)(46)(47)(48)(49)(50), which serves a single task, i.e. to establish the vital physical interaction that promotes ATP hydrolysis in Hsp70 (17,18,(23)(24)(25)(26) , thus allowing the chaperone to capture client substrates (19). The proper docking of the J-domain onto ATPbound Hsp70 is ensured by a core set of intermolecular contacts occurring along the helix II--loop (HDP motif)--helix III region of the J-domain, which is highly conserved from bacteria to human (23,24).…”

Structural influence of the conserved Hsp40 HPD tripeptide on Hsp70 chaperone function

“…The D35-R167 bond has been previously reported to be a necessary interaction between DnaK and Jd (6). Recent work has suggested the need for conserving D35-R167 contact in Hsp40-Hsp70 systems across different species (4). This need may stem from the requirement to link J-domain association with the induction of ATPase activity in DnaK.…”

“…These may represent a shift in the binding character of the J-domain with the H33Q mutation. Previous work has suggested the possibility for a range of interaction modes to be employed in Hsp40-Hsp70 systems across different species, provided that the key residue contacts are maintained (4). Disruption of these key interactions, as observed with altered bond propensities may contribute to the inability of the mutants to induce ATPase activity and DnaK function.…”

“…In the ATP bound state, the linker has been observed to bind the NBD facilitating the docked conformation (2,3). Disruption of the NBD-linker interaction has been suggested to promote the undocked conformation associated with ATP-hydrolysis (4). Exchanging the bound ADP with ATP returns the chaperone into the open configuration which restarts the cycle (5).…”