Chaperones are the molecular machines tasked with maintaining cellular homeostasis by aiding nascent peptide folding, promoting substrate translocation into organelles, and preventing aggregation by unfolding misfolded conformers. A target in the prevention of neurodegenerative diseases, heat shock protein (70 kDa), Hsp70, is a prevalent, conserved chaperone that is more highly expressed in stressed and aging cells. Comprising a nucleotide binding domain (NBD) and substrate binding domain (SBD), we investigated its mechanical properties in both domains separately with laser optical tweezers and corresponding coarse-grained pulling molecular dynamics simulations. We discovered a hierarchical shift in the mechanical stability from lobe I to lobe II in the NBD and disproved previous hypotheses that the interdomain linker was the means of allosteric communication between the NBD and SBD. In the SBD, fluctuations in our experiments revealed mechanical communication across the connecting loop between the alpha helical lid and beta sandwich subdomains. Stabilization in the latter beta sheet induces folding and hinge formation in Helix A and B of the lid subdomain after hydrolysis in the NBD. These insights clarify Hsp70 mechanics, have a larger impact for understanding mechanisms of other ATPases in the Actin/sugar kinase superfamily, and help us understand how substrate binding and release occurs during assisted protein folding.

Hsp70’s domains alternating flexibilities enable its chaperone action

254th National Meeting (2017)

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