Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling.

In Escherichia coli, DnaK is essential for the replication of bacteriophage lambda DNA; this in vivo activity provides the basis of a screen for mutations affecting DnaK function. Mn PCR was used to introduce mutations into residues 405-468 of the C-terminal polypeptide-binding domain of DnaK. These mutant proteins were screened for the ability to propagate bacteriophage lambda in the background of a dnaK deficient cell line, BB1553. This initial screen identified several proteins which were mutant at multiple positions. The multiple mutants were further dissected into single mutants which remained negative for lambda propagation. Four of these single-site mutants were purified and assayed for biochemical functionality. Two single-site mutations, F426S and S427P, are localized in the peptide binding site and display weakened peptide binding affinity. This indicates that the crystallographically determined peptide binding site is also critical for in vivo lambda replication. Two other mutations, K414I and N451K, are located at the edge of the beta-sandwich domain near alpha-helix A. The K414I mutant binds peptide moderately well, yet displays defects in allosteric functions, including peptide-stimulated ATPase activity, ATP-induced changes in tryptophan fluorescence, ATP-induced peptide release, and elevated ATPase activity. The K414 position is close in tertiary structure to the linker region to the ATPase domain and reflects a specific area of the peptide-binding domain which is necessary for interdomain coupling. The mutant N451K displays defects in both peptide binding and allosteric interaction. Copyright 1999 Academic Press.