Modulation of substrate specificity of the DnaK chaperone by alteration of a hydrophobic arch.
Hsp70 chaperones assist protein folding by reversible interaction with extended hydrophobic segments of substrate polypeptides. We investigated the contribution of three structural elements of the substrate- binding cavity of the Escherichia coli homologue, DnaK, to substrate specificity by investigating mutant DnaK proteins for binding to cellulose-bound peptides. Deletion of the C-terminal subdomain (Delta539-638) and blockage of the access to the hydrophobic pocket in the substrate-binding cavity (V436F) did not change the specificity, although the latter exchange reduced the affinity to all peptides investigated. Mutations (A429W, M404A/A429W) that affect the formation of a hydrophobic arch spanning over the bound substrate disfavored DnaK binding, especially to peptides with short stretches of consecutive hydrophobic residues flanked by acidic residues, while binding to most other peptides remained unchanged. The arch thus contributes to the substrate specificity of DnaK. This finding is of particular interest, since of all the residues of the substrate-binding cavity that contact bound substrate, only the arch-forming residues show significant variation within the Hsp70 family.
Rüdiger
S
S
Institut für Biochemie und Molekularbiologie, Hermann-Herder-Str. 7, Universität Freiburg, D-79104, Germany.
Mayer
M P
MP
Schneider-Mergener
J
J
Bukau
B
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; C063842:dnaK protein, E coli