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Conformational equilibria in allosteric control of Hsp70 chaperones.

Molecular cell (2021-08-29)
Wei Wang, Qinglian Liu, Qun Liu, Wayne A Hendrickson
ABSTRACT

Heat-shock proteins of 70 kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Adenosine 5′-diphosphate monopotassium salt dihydrate, bacterial, ≥95%, powder
Sigma-Aldrich
Rosetta(DE3) Competent Cells - Novagen, Rosetta host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli.
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, 99%