# Mechanism of Hsp70 activation: How J-domain proteins push for ATP hydrolysis

## TL;DR

This study reveals how J-domain proteins activate Hsp70 chaperones by pushing structural changes that promote ATP hydrolysis.

## Contribution

The paper provides a mechanistic model of how J-domain proteins induce ATP hydrolysis in Hsp70 through steric and allosteric effects.

## Key findings

- JD binding rearranges the Hsp70 nucleotide-binding pocket into a hydrolysis-competent state via T199-ATP contact.
- Allosteric signals propagate through β-strands 13 and 14, driven by steric repulsion between JD helix III and the SBD.
- Structural changes induced by JDP binding reposition conserved residues to facilitate ATP hydrolysis.

## Abstract

Hsp70 chaperones are crucial for maintaining protein homeostasis by regulating the stability and conformational states of client polypeptides through ATP dependent cycles of binding and release. These cycles are driven by conformational transitions in Hsp70 upon ATP binding and hydrolysis. The ATPase activity of Hsp70 is controlled by J-domain protein (JDP) cochaperones, which allosterically stimulate ATP hydrolysis through interactions between their J-domains (JDs) and Hsp70. The JD binds at the interface between the nucleotide binding domain (NBD) and substrate binding domain (SBD) of ATP bound Hsp70. While it is well-established that JD interaction involves the conserved histidine-proline-aspartic acid (HPD) motif and residues in helices II and III, the mechanism by which JD-induced allosteric signals propagate to the distal nucleotide-binding pocket - and the conformational changes that facilitate ATP hydrolysis - remains unclear. Here, we addressed these questions using all-atom free energy simulations and dynamic network analysis, starting from crystal structures of ATP-bound DnaK (Hsp70) alone and in complex with the JD of DnaJ (JDP). We show that JD binding rearranges the NBD nucleotide-binding pocket into a hydrolysis competent state, characterized by the formation of a contact between the hydroxyl group of the universally conserved threonine 199 (T199) and the γ-phosphate of ATP. Network analysis revealed that the allosteric signal driving this rearrangement propagates along β-strands 13 and 14 towards T199. Moreover, we provide a mechanistic understanding for this signal transmission, demonstrating that steric repulsion between JD helix III and the SBD, alongside β-strand 14 disruption, facilitate T199-ATP contact formation. Overall, our study provides mechanistic insights into allosteric signal transmission within Hsp70, bridging the gap between JD binding and ATPase stimulation.

Hsp70 chaperones, together with J‑domain protein (JDP) co-chaperones, play a central role in maintaining protein homeostasis across diverse cell types and subcellular compartments. By coupling ATP binding and hydrolysis to conformational change, Hsp70s assist in protein folding, trafficking, degradation, and protein-protein interactions. Despite extensive study, the molecular basis by which JDPs stimulate the ATPase activity of Hsp70 has remained a challenging question. Here, using the crystal structure of Hsp70 bound to the J-domain of its JDP partner, we performed molecular dynamic simulations and complementary thermodynamic analyses to elucidate how JDP binding reshapes Hsp70 to promote ATP hydrolysis. Our results show that JDP binding generates a “steric push” that initiates an allosteric signal cascade, propagated through coordinated global and local conformational rearrangements. These structural changes reposition a conserved threonine residue closer to the ATP molecule, thereby facilitating catalysis. Our findings establish a structural and energetic framework linking JDP binding to the stimulation of Hsp70 ATP hydrolysis, providing mechanistic insight into a central step in chaperone function.