Modulation of substrate specificity of the DnaK chaperone by alteration ofa hydrophobic arch

Hsp70 chaperones assist protein folding by reversible interaction with exte nded hydrophobic segments of substrate polypeptides. We investigated the co ntribution of three structural elements of the substrate-binding cavity of the Escherichia coli homologue, DnaK, to substrate specificity by investiga ting mutant DnaK proteins for binding to cellulose-bound peptides. Deletion of the C-terminal subdomain (Delta 539-638) and blockage of the access to the hydrophobic pocket in the substrate-binding cavity (V436F) did not chan ge the specificity, although the latter exchange reduced the affinity to al l peptides investigated. Mutations (A429W, M404A/A429W) that affect the for mation of a hydrophobic arch spanning over the bound substrate disfavored D naK binding, especially to peptides with short stretches of consecutive hyd rophobic residues flanked by acidic residues, while binding to most other p eptides remained unchanged. The arch thus contributes to the substrate spec ificity of DnaK. This finding is of particular interest, since of all the r esidues of the substrate-binding cavity that contact bound substrate, only the arch-forming residues show significant variation within the Hsp70 famil y. (C) 2000 Academic Press.