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

金黃色葡萄球菌所攜帶的質體pI258上的cadA基因所表現的CadA蛋白，提供細菌對於鎘離子的抗性。而大腸桿菌基因copA，則是提供細菌對於一價銅離子的抗性。CadA蛋白和CopA蛋白，都屬於P-type ATPase中的CPx-type ATPase。這類運輸金屬的膜蛋白，具有一些序列及結構上的特徵，像是在蛋白的N端上皆具有Cys-X-X-Cys序列，並且在第六個穿膜區域，具有Cys-Pro-X motif。本論文研究的目的，就是以直接的生化方法，探討這些CPx-type ATPase結構上所特有的區域。首先利用定點突變的方法，將CadA蛋白N端第23、26個位置的cysteine，突變成serine及glycine，所得到的結果顯示，這些CadA cysteine被置換的細菌，對於鎘離子的抗性明顯下降。而利用細胞堆積鎘離子分析實驗，來測量突變的CadA蛋白其排放鎘離子的能力是否受影響。本論文研究中發現，這些CadA cysteine被置換的細菌，其排放鎘離子的能力也下降了。為進一步探討CPx-type ATPases的受質特異性、與CXXC序列的相關性。在本研究中，利用雜交蛋白的方法，將CadA及CopA兩種功能迥異的CPx-type ATPases，進行部分區域置換的雜交蛋白。本論文研究結果發現，CXXC對於CopA蛋白可能不具篩選特異性受質的角色，而受質特異性的決定位置，可能在CopA蛋白的其他位置。然而，對於CadA蛋白，CXXC在受質特異性方面，可能具備某些重要性的角色，而CXXC以及所在的N端蛋白區域，也可能是扮演調節性角色，對於執行CadA功能相當重要。

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.