利用 CadA/CopA雜交蛋白模式來探討CPx-ATPase功能性結構的生化特性

Abstract

Staphylococcal plasmid pI258 carries cadA gene for cadmium resistance. A similar gene was also found in E. coli chromosomal mediated copper resistance called copA . Both gene products, the CadA and CopA are members of cation-transporting P-type ATPases, and recently re-grouped into the CPx-type ATPase family, which are characterized by some unique features including the N-terminal Cys-X-X-Cys motif as well as the 6th-transmembrane-buried Cys-Pro-X motif. In this study, we have designed experiments to elucidate the functional roles of these motifs shared by all CPx-type ATPases. Using site-directed mutagenesis approaches, the cysteine residues within the CXXC motif of CadA were replaced by either serine or glycine residue at the amino acid positions 23 and 26. These cysteine-substituted mutants of CadA showed reductions in cadmium resistance when transformed them into cadmium-sensitive E. coli RW3110. Uptake assays using isotopic cadmium have also demonstrated these cysteine-substituted mutants of CadA accumulated more cadmium than wild-type CadA, suggesting the CXXC might be important for the cadmium transport activity. In order to determine the role of CXXC in substrate-specificity, a chimera protein strategy was taken to create hybrid proteins with N-terminal portion of CadA and C-terminal portion of CopA or vise versa. Our results have shown that CXXC might play different role in CadA and CopA. In CopA, the CXXC alone did not determine its substrate-specificity. And the copper-specificity of CopA might rely on the different location of that protein, however the CXXC might play a supporting role for the function. On the other hand, the CXXC motif in CadA might be more important for its substrate-specificity as well as alone with the N-terminal domain might also play a regulatory role in CadA activity.