SUBSTRATE-SPECIFICITY OF THE DNAK CHAPERONE DETERMINED BY SCREENING CELLULOSE-BOUND PEPTIDE LIBRARIES

Hsp70 chaperones assist protein folding by ATP-dependent association w ith linear peptide segments of a large variety of folding intermediate s. The molecular basis for this ability to differentiate between nativ e and non-native conformers was investigated for the DnaK homolog of E scherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues, In the fo lded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic co re of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic resi dues. Acidic residues are excluded from the core and disfavored in fla nking regions, The energetic contribution of all 20 amino acids for Dn aK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences wit h high accuracy.