Publication

Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein

van der Wolk, J. P. W., Klose, M., de Wit, J., Blaauwen, T. D., Freudl, R. & Driessen, A. J. M., 11-Aug-1995, In : The Journal of Biological Chemistry. 270, 32, p. 18975-18982 8 p.

Research output: Contribution to journalArticleAcademicpeer-review

APA

van der Wolk, J. P. W., Klose, M., de Wit, J., Blaauwen, T. D., Freudl, R., & Driessen, A. J. M. (1995). Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein. The Journal of Biological Chemistry, 270(32), 18975-18982. https://doi.org/10.1074/jbc.270.32.18975

Author

van der Wolk, J.P.W. ; Klose, M ; de Wit, Janny ; Blaauwen, T.den ; Freudl, R ; Driessen, A.J.M. / Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein. In: The Journal of Biological Chemistry. 1995 ; Vol. 270, No. 32. pp. 18975-18982.

Harvard

van der Wolk, JPW, Klose, M, de Wit, J, Blaauwen, TD, Freudl, R & Driessen, AJM 1995, 'Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein', The Journal of Biological Chemistry, vol. 270, no. 32, pp. 18975-18982. https://doi.org/10.1074/jbc.270.32.18975

Standard

Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein. / van der Wolk, J.P.W.; Klose, M; de Wit, Janny; Blaauwen, T.den; Freudl, R; Driessen, A.J.M.

In: The Journal of Biological Chemistry, Vol. 270, No. 32, 11.08.1995, p. 18975-18982.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

van der Wolk JPW, Klose M, de Wit J, Blaauwen TD, Freudl R, Driessen AJM. Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein. The Journal of Biological Chemistry. 1995 Aug 11;270(32):18975-18982. https://doi.org/10.1074/jbc.270.32.18975


BibTeX

@article{e4d7ecf0bcad4f7baeec35b55dbeebf0,
title = "Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein",
abstract = "The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the ''so-called'' Walker B-motif, hXhhD (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg2+-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coil SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth complement an E. coil secA amber mutant strain, and the E. coil SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg2+-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp 215 are involved in the binding of the Mg2+-ion when Mg2+-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.",
keywords = "TRANSLOCATION ATPASE, PLASMA-MEMBRANE, ESSENTIAL COMPONENT, PRECURSOR PROTEINS, CRYSTAL-STRUCTURE, TRIGGER FACTOR, PRO-OMPA, EXPORT, GENE, INVIVO",
author = "{van der Wolk}, J.P.W. and M Klose and {de Wit}, Janny and T.den Blaauwen and R Freudl and A.J.M. Driessen",
note = "Journal UG 11 N954 BIOL CHEM",
year = "1995",
month = "8",
day = "11",
doi = "10.1074/jbc.270.32.18975",
language = "English",
volume = "270",
pages = "18975--18982",
journal = "The Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC",
number = "32",

}

RIS

TY - JOUR

T1 - Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein

AU - van der Wolk, J.P.W.

AU - Klose, M

AU - de Wit, Janny

AU - Blaauwen, T.den

AU - Freudl, R

AU - Driessen, A.J.M.

N1 - Journal UG 11 N954 BIOL CHEM

PY - 1995/8/11

Y1 - 1995/8/11

N2 - The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the ''so-called'' Walker B-motif, hXhhD (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg2+-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coil SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth complement an E. coil secA amber mutant strain, and the E. coil SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg2+-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp 215 are involved in the binding of the Mg2+-ion when Mg2+-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

AB - The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the ''so-called'' Walker B-motif, hXhhD (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg2+-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coil SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth complement an E. coil secA amber mutant strain, and the E. coil SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg2+-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp 215 are involved in the binding of the Mg2+-ion when Mg2+-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

KW - TRANSLOCATION ATPASE

KW - PLASMA-MEMBRANE

KW - ESSENTIAL COMPONENT

KW - PRECURSOR PROTEINS

KW - CRYSTAL-STRUCTURE

KW - TRIGGER FACTOR

KW - PRO-OMPA

KW - EXPORT

KW - GENE

KW - INVIVO

U2 - 10.1074/jbc.270.32.18975

DO - 10.1074/jbc.270.32.18975

M3 - Article

VL - 270

SP - 18975

EP - 18982

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

SN - 0021-9258

IS - 32

ER -

ID: 1217415