SecA Dimer Research Articles

The Carboxyl-Terminal Region Is Essential for Sec-A Dimerization

SecA, comprising 901 amino acid residues, exists as a dimer. By means of size exclusion chromatography and chemical cross-linking analysis, five truncated SecA derivatives were examined to identify the region of SecA essential for dimer formation. Among them, only N95 (Δ832-901) retained SecA activity. N95 existed as a dimer, indicating that the carboxyl-terminal three cysteine residues are dispensable for physiological dimerization. Both N76 (Δ675-901) and N66 (Δ583-901) existed as monomers. Monomeric N76 was able to bind to ATP, indicating that the dimerization of SecA is not a prerequisite for ATP binding. However, the rate of ATP hydrolysis by N76 was 25% of that by SecA. C53 (Δ1-437) and C28 (Δ1-661) formed dimers irrespective of the presence or absence of 2-mercaptoethanol. C28, but not C53, also existed as an oligomer in the absence of 2-mercaptoethanol, suggesting that the 438-661 region present in C53 prevents intermolecular disulfide bond formation at the carboxyl-terminal cysteine residue. From these results, the region essential for the physiological dimer formation was concluded to be located in the 662-831 region of SecA.

Site-specific proteolysis of the Escherichia coli SecA protein in vivo.

A seven-amino-acid cleavage site specific for tobacco etch virus (TEV) protease was introduced into SecA at two separate positions after amino acids 195 and 252. Chromosomal wild-type secA was replaced by these secA constructs. Simultaneous expression of TEV protease led to cleavage of both SecA derivatives. In the functional SecA dimer, proteolysis directly indicated surface exposure of the TEV protease cleavage sites. Cleavage of SecA near residue 195 generated an unstable proteolysis product and a secretion defect, suggesting that this approach could be used to inactivate essential proteins in vivo.

Identification of the Magnesium-binding Domain of the High-affinity ATP-binding Site of the Bacillus subtilis and Escherichia coli SecA Protein

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 Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli 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 Mg(2+)-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 Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in [formula omitted][formula omitted

SecA is an essential component of the protein secretory machinery of Escherichia coli . SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis. The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structure. It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation. SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution. Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol. It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.