Investigating the Determinants of Substrate Binding Orientation within the E. coli Hsp70 Chaperone Protein

Abstract

Molecular chaperone proteins play a critical role in maintenance of the cell’s proteome through protein quality control. Heat shock protein 70 chaperones (Hsp70s) are an evolutionarily conserved and diverse family of chaperone proteins that assist with proper protein folding and prevention of aggregation in the cell. Previous research has shown that the substrate‐binding domain of the E. coli Hsp70, DnaK, contains binding pockets of low stringency extending bilaterally from a highly hydrophobic central pocket. The SBD interacts with short hydrophobic sequences of substrates by “reading” the sequence. The SBD will then bind the substrate in one of two orientations: an N‐C, or forward, orientation, or a C‐N, reverse, orientation. Research done thus far on substrate binding has not revealed an exact mechanism that determines orientation. In this study, peptides possessing the same sequence with opposing backbone directions were used to test the significance of backbone interactions with the SBD as well as residue side chains and their interactions with the SBD. The analysis of the bound orientation of these peptides is accomplished by disulfide cross‐linking using peptides containing an N‐terminal Cys and SBD proteins with a Cys introduced in such a way where it can be reached in only one orientation. The results indicated slight energetic favorable biases for the interactions of residue side chains and the SBD pockets. The results also place a high importance on leucine residues in substrates as a determinant of optimal pocket ‐ residue fit and binding orientation. Altogether, the data suggest that binding occurs in the orientation that supports the most optimal fit of residue side chains into binding pockets, which reveals further intricacies and selectivity involved in substrate binding for Hsp70s.