Model Peptides Enable Comparisons of Substrate Binding Modes of Hsp70 Molecular Chaperones

Hsp70 molecular chaperones play a wide array of essential roles in the cell by exploiting their ability to bind incompletely folded client proteins. Our past study of the Escherichia coli Hsp70, DnaK, bound to short peptides revealed many details of the interaction between a model substrate and the "pockets" of the canonical binding cleft within the chaperone substrate binding domain (SBD). A major finding was that peptides bound the SBD cleft in either N- to C- or C- to N- orientations with nearly equal frequencies. The current study asks key questions: What determines preferred orientation in the bound substrate? Also, are the binding behaviors observed for DnaK also found for mammalian homologues? Here, we use NMR and cross-linking methods developed in our past study and compare modes of binding to DnaK and the two major human cytoplasmic Hsp70s, Hsc70 and HspA1. Model peptides examined include a "palindromic" peptide, central residue variant peptides, and examples of naturally occurring Hsp70 clients. The results reveal that DnaK is agnostic to backbone orientation of the bound peptide, while Hsc70 modestly favors the C- to N- orientation, and HspA1 strongly favors the N- to C- orientation. Moreover, substrate affinities in general are similar for DnaK and Hsc70 and weaker for HspA1. The binding energy landscapes of these chaperones also differ, as the data suggest deeper energy wells for bound states in DnaK and HspA1 and a more rugged landscape for Hsc70. We speculate that these properties enable the chaperones to perform their physiological functions more effectively.