Specificity of DnaK for arginine/lysine and effect of DnaJ on the amino acid specificity of DnaK.

Molecular chaperones form a class of proteins that bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides and are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. Chaperones70, including the DnaK chaperone of Escherichia coli, interact specifically with peptides enriched in internal hydrophobic residues, with a preference for positively charged peptides. We previously reported that DnaK interacts with the hydrophobic amino acids Ile, Leu, Val, Ala, Phe, Trp, and Tyr. In the present study, we show that DnaK also possesses a specific binding site for the positively charged amino acids arginine and lysine. Furthermore, the binding of arginine and lysine to DnaK is strengthened when its hydrophobic binding sites are occupied. The specificity of DnaK for Arg/Lys is supported by DnaK-peptide binding studies; the homopolypeptides poly-Arg and poly-Lys interact with DnaK, contrasting with other hydrophilic homopolypeptides, and hydrophobic peptides interact more strongly with DnaK if they contain Arg/Lys at their N terminus. Interestingly, the cochaperone DnaJ attenuates the interaction of DnaK with hydrophobic amino acids while strengthening its interaction with arginine or lysine. The interaction of DnaK with both hydrophobic sequences and with arginine and lysine, and its modulation by DnaJ, may have important implications in both protein folding and protein insertion into membranes.