TY - JOUR
T1 - Mutations in the substrate binding domain of the Escherichia coli 70 kDa molecular chaperone, DnaK, which alter substrate affinity or interdomain coupling
AU - Montgomery, Diana L.
AU - Morimoto, Richard I.
AU - Gierasch, Lila M.
N1 - Funding Information:
The authors thank Hwa-Ping Feng and Joanna Feltham for helpful discussions and critical readings of the manuscript; Huira Chung for her preliminary work on the project; Roger McMacken for advice, suggestions, and co-chaperone proteins; Robert and Ruby McDonald for the use of their Alpha Scan Fluorometer; Jonathan Widom for the use of his reverse-phase HPLC system; Linda Rotondi for synthesizing peptide NRW4; and Robert Weis for the use of his DSC. This work was supported by a National Research Service Award GM16719 (D.L.M.) and grants GM38109 (R.I.M.) and GM27616 (L.M.G.) from the National Institutes of Health.
PY - 1999/2/26
Y1 - 1999/2/26
N2 - In Escherichia coli, DnaK is essential for the replication of bacteriophage λ 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 λ 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 λ 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 λ replication. Two other mutations, K414I and N451K, are located at the edge of the β-sandwich domain near α-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.
AB - In Escherichia coli, DnaK is essential for the replication of bacteriophage λ 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 λ 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 λ 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 λ replication. Two other mutations, K414I and N451K, are located at the edge of the β-sandwich domain near α-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.
KW - Allosterism
KW - Calorimetry
KW - Chaperones
KW - DnaK
KW - Fluorescence
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U2 - 10.1006/jmbi.1998.2514
DO - 10.1006/jmbi.1998.2514
M3 - Article
C2 - 10024459
AN - SCOPUS:0033605086
SN - 0022-2836
VL - 286
SP - 915
EP - 932
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -