Abstract

The 70-kDa heat shock protein (Hsp70) interacts with the polypeptide segments of abundant native proteins to fulfill various cellular activities in both stress and normal conditions. However, a non-native linear polypeptide NR (NRLLLTG) is widely used for the study of Hsp70 substrate binding properties, which is too simple to reflect the complex status of Hsp70 substrates in living organisms. To further broaden our knowledge in this area, we screened 2645 polypeptides derived from 78 biologically relevant proteins and identified eight native peptide substrates (named VP1[~]VP8) for bacterial Hsp70 DnaK. Consistent with previous findings, the amino acid distribution in VP1[~]VP8 were enriched in aliphatic and basic residues, and most of their residues were buried in folded proteins as well. Besides, the substrate binding properties for seven polypeptides were largely the same as observed in NR, suggesting their conserved binding mode to DnaK. However, VP5, which contains more percentage of positively charged residues, demonstrates divergent substrate binding properties during in-vitro biochemical studies. Moreover, VP5 efficiently inhibits the refolding activity of DnaK and bacterial viability, implying its potential to be a good lead peptide for antibacterial drug development.