Exploring Hsp70 chaperone interactions with a full‐length substrate

Abstract

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding and preventing proteins from aggregation (Clérico et al., 2015). Despite extensive research that reveals a selective promiscuity between the Hsp70 substrate‐binding domain (SBD) and client proteins, there is a paucity of data on the interaction of Hsp70s with large protein clients. To investigate the interaction between Hsp70s and a full‐length substrate, we chose to characterize the binding of the E. coli homolog,DnaK, to the precursor of alkaline phosphatase (proPhoA). proPhoA is an endogenous substrate of DnaK that is unfolded and soluble under reducing conditions, and contains at least five sequences that bind DnaK when presented as short peptides (Rüdiger et al., 1997; Wild et al., 1992). To simplify the experimental conditions we used truncated versions of proPhoA (~100 residues long) that contain 1 or 2 binding sites. We used biophysical techniques to reveal the affinity of binding, and NMR spectroscopy to elucidate the mode of chaperone binding to the proPhoA fragments. We found that there is a hierarchy of affinities among the 5 potential binding sites, and novel cryptic binding sites have been observed. In addition, data suggest there may be a role for residues beyond the canonical five‐residue core sequences. The mechanism of DnaK binding site selection on a substrate containing multiple potential sites and the structural basis of interaction between the chaperone and its respective binding sites on a full‐length substrate will be described.Support or Funding InformationNIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/2021