HSPA1A conformational mutants reveal a conserved structural unit in Hsp70 proteins

Biochim Biophys Acta Gen Subj. 2020 Jan;1864(1):129458. doi: 10.1016/j.bbagen.2019.129458. Epub 2019 Oct 30.


Background: The Hsp70 proteins maintain proteome integrity through the capacity of their nucleotide- and substrate-binding domains (NBD and SBD) to allosterically regulate substrate affinity in a nucleotide-dependent manner. Crystallographic studies showed that Hsp70 allostery relies on formation of contacts between ATP-bound NBD and an interdomain linker, accompanied by SBD subdomains docking onto distinct sites of the NBD leading to substrate release. However, the mechanics of ATP-induced SBD subdomains detachment is largely unknown.

Methods: Here, we investigated the structural and allosteric properties of human HSPA1A using hydrogen/deuterium exchange mass spectrometry, ATPase assays, surface plasmon resonance and fluorescence polarization-based substrate binding assays.

Results: Analysis of HSPA1A proteins bearing mutations at the interface of SBD subdomains close to the interdomain linker (amino acids L399, L510, I515, and D529) revealed that this region forms a folding unit stabilizing the structure of both SBD subdomains in the nucleotide-free state. The introduced mutations modulate HSPA1A allostery as they localize to the NBD-SBD interfaces in the ATP-bound protein.

Conclusions: These findings show that residues forming the hydrophobic structural unit stabilizing the SBD structure are relocated during ATP-activated detachment of the SBD subdomains to different NBD-SBD docking interfaces enabling HSPA1A allostery.

General significance: Mutation-induced perturbations tuned HSPA1A sensitivity to peptide/protein substrates and to Hsp40 in a way that is common for other Hsp70 proteins. Our results provide an insight into structural rearrangements in the SBD of Hsp70 proteins and highlight HSPA1A-specific allostery features, which is a prerequisite for selective targeting in Hsp-related pathologies.

Keywords: Allostery; Folding; Heat-shock protein 70; Molecular chaperones; Mutation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / genetics*
  • Allosteric Regulation / genetics*
  • Binding Sites / genetics
  • Deuterium Exchange Measurement
  • HSP70 Heat-Shock Proteins / chemistry
  • HSP70 Heat-Shock Proteins / genetics*
  • Humans
  • Mutation / genetics
  • Protein Binding / genetics
  • Protein Conformation*
  • Protein Domains / genetics


  • HSP70 Heat-Shock Proteins
  • HSPA1A protein, human
  • Adenosine Triphosphate