Ramaswamy S, Kaur P
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA.
J Biol Chem. 1998 Apr 10;273(15):9243-8. doi: 10.1074/jbc.273.15.9243.
ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.
砷转运ATP酶A亚基（ArsA蛋白）是大肠杆菌中质粒编码的阴离子转运ATP酶的催化成分，它含有两个共有核苷酸结合结构域A1和A2，由一个柔性连接子相连。先前研究表明，ATP在紫外线激活后能与A1结构域交联，但不与A2结构域交联。ATP类似物5'-对氟磺酰苯甲酰腺苷（FSBA）被用于探测ArsA的核苷酸结合结构域。对共价标记的蛋白进行部分胰蛋白酶消化，然后用抗FSBA血清对片段进行蛋白质印迹分析。标记片段的N端氨基酸序列表明，在有无亚锑酸盐的情况下，FSBA都优先结合C端结构域A2。通过对胰蛋白酶消化的保护作用来确定两个核苷酸结合位点的占据情况。胰蛋白酶在连接子中的Arg290处切割ArsA蛋白，产生一个32 kDa的N端片段和一个27 kDa的C端片段。32 kDa的片段结构紧密，胰蛋白酶基本无法接近；然而，27 kDa的片段会被进一步切割。与结合C端结构域的FSBA孵育，会使27 kDa的片段得到显著保护。单独与ATP孵育时，该片段不受保护，这表明A2可能未被占据。然而，与ATP和亚锑酸盐一起孵育时，几乎能完全保护其不被胰蛋白酶切割。ATP和FSBA共同模拟了ATP和亚锑酸盐的作用，这意味着这种完全受保护的构象可能是两个位点都被核苷酸占据的结果。有人提出，ArsA中的A1位点是一个高亲和力的ATP位点，而异构效应配体亚锑酸盐是使ATP结合到A2所必需的，从而导致催化协同作用。因此，亚锑酸盐的结合可能作为一个开关来调节ATP与A2的结合，进而调节ArsA的ATP酶活性。
