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Author(s): VANDERWOLK JPW; KLOSE M; DEWIT JG; DENBLAAUWEN T; FREUDL R; DRIESSEN AJM
Title: IDENTIFICATION OF THE MAGNESIUM-BINDING DOMAIN OF THE HIGH-AFFINITY ATP BINDING-SITE OF THE BACILLUS-SUBTILIS AND ESCHERICHIA-COLI SECA PROTEIN
Source: JOURNAL OF BIOLOGICAL CHEMISTRY 270 (32): 18975-18982
Date: 1995 AUG 11
Document Type: Journal : Article
DOI:
Language: English
Comment:
Address: UNIV GRONINGEN,DEPT MICROBIOL,9751 NN HAREN,NETHERLANDS.
UNIV GRONINGEN,GRONINGEN BIOMOLEC SCI & BIOTECHNOL INST,9751 NN HAREN,NETHERLANDS. KFA JULICH GMBH,FORSCHUNGSZENTRUM,INST BIOTECHNOL 1,D-52425 JULICH,GERMANY. Reprint:
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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.
Cited References: ABRAHAMS JP, 1994, NATURE, V370, P621 AKITA M, 1991, BIOCHEM BIOPH RES CO, V174, P211 AMANN E, 1988, GENE, V69, P301 BERCHTOLD H, 1993, NATURE, V365, P126 BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248 BREUKINK E, 1992, BIOCHEMISTRY-US, V31, P1119 BRUNDAGE L, 1990, CELL, V62, P649 CABELLI RJ, 1988, CELL, V55, P683 CASADABAN MJ, 1976, J MOL BIOL, V104, P541 CHANG CN, 1978, P NATL ACAD SCI USA, V75, P361 CROOKE E, 1987, P NATL ACAD SCI USA, V84, P5216 CROOKE E, 1988, EMBO J, V7, P1831 CUNNINGHAM K, 1989, EMBO J, V8, P955 CUNNINGHAM K, 1989, P NATL ACAD SCI USA, V86, P8630 DEVRIJE T, 1987, BIOCHIM BIOPHYS ACTA, V900, P63 DRIESSEN AJM, 1993, BIOCHEMISTRY-US, V32, P13190 DRIESSEN AJM, 1994, J MEMBRANE BIOL, V142, P145 ECONOMOU A, 1994, CELL, V78, P835 FLAHERTY KM, 1994, J BIOL CHEM, V269, P12899 FREUDL R, 1985, EMBO J, V4, P3593 HARTL FU, 1990, CELL, V63, P269 HOWARD JB, 1994, ANNU REV BIOCHEM, V63, P235 KIM YJ, 1994, CELL, V78, P845 KLOSE M, 1993, J BIOL CHEM, V268, P4504 KOONIN EV, 1993, FEBS LETT, V298, P6 KUNKEL TA, 1985, P NATL ACAD SCI USA, V82, P488 LAEMMLI UK, 1970, NATURE, V227, P680 LAKOWICZ JR, 1986, PRINCIPLES FLUORESCE LANDT O, 1990, GENE, V96, P125 LANZETTA PA, 1979, ANAL BIOCHEM, V100, P95 LILL R, 1989, EMBO J, V8, P961 LILL R, 1990, CELL, V60, P271 MATSUYAMA S, 1990, J BIOL CHEM, V265, P8760 MEAD DA, 1986, PROTEIN ENG, V1, P67 MILLER JH, 1972, EXPT MOL GENETICS MITCHELL C, 1994, MOL MICROBIOL, V10, P483 MULLER CW, 1992, J MOL BIOL, V224, P159 OLIVER DB, 1981, CELL, V25, P765 OLIVER DB, 1982, CELL, V30, P311 OVERHOFF B, 1991, MOL GEN GENET, V228, P417 PAI EF, 1990, EMBO J, V9, P2351 SADAIE Y, 1991, GENE, V98, P101 SAMBROOK J, 1989, MOL CLONING LABORATO SCHIEBEL E, 1991, CELL, V64, P927 SCHMIDT MG, 1988, J BACTERIOL, V170, P3404 SCHMIDT MG, 1989, J BACTERIOL, V171, P643 SHINKAI A, 1991, J BIOL CHEM, V266, P5824 STORY RM, 1992, NATURE, V355, P374 TAKAMATSU H, 1992, J BACTERIOL, V174, P4308 VANDERWOLK J, 1993, MOL MICROBIOL, V8, P31 WALKER JE, 1982, EMBO J, V1, P945 WARNER HR, 1978, NATURE, V272, P32 WEISS JB, 1988, P NATL ACAD SCI USA, V85, P8978 WICKNER W, 1991, ANNU REV BIOCHEM, V60, P101 WILBANKS SM, 1994, J BIOL CHEM, V269, P12893 YANISCHPERRON C, 1985, GENE, V33, P103 |