51. HSPA1A conformational mutants reveal a conserved structural unit in Hsp70 proteins
- Author
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Petr Müller, Petr Man, Josef Houser, Filip Trčka, Pavla Vankova, Daniel Kavan, Veronika Vandová, and Michal Durech
- Subjects
Protein Conformation ,Allosteric regulation ,Biophysics ,Peptide ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Adenosine Triphosphate ,Allosteric Regulation ,Protein Domains ,Humans ,HSP70 Heat-Shock Proteins ,Surface plasmon resonance ,Molecular Biology ,Structural unit ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,Chemistry ,Deuterium Exchange Measurement ,Docking (molecular) ,Proteome ,Mutation ,Hydrogen–deuterium exchange ,Linker ,030217 neurology & neurosurgery ,Protein Binding - Abstract
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.
- Published
- 2019