Structural determinants of HscA peptide-binding specificity

The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.