MUTATIONS IN THE C-TERMINAL FRAGMENT OF DNAK AFFECTING PEPTIDE BINDING

Escherichia coli DnaK acts as a molecular chaperone through its ATP-re gulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing th e polypeptide substrate binding domain is lethal in wild-type E. coli, This dominant-negative phenotype may result from the nonproductive bi nding of CTF to cellular polypeptide targets of DnaK, Mutations affect ing DnaK substrate binding were identified by selecting noncytotoxic C TF mutants followed by in vitro screening, The clustering of such muta tions in the three-dimensional structure of CTF suggests the model tha t loops L(1,2) and L(4,5) form a rigid core structure critical for int eractions with substrate.