Zhong X, Tai P C
Department of Biology, Georgia State University, Atlanta 30303, USA.
J Bacteriol. 1998 Mar;180(6):1347-53. doi: 10.1128/JB.180.6.1347-1353.1998.
The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg2+ and Mn2+ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37 degrees C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10 degrees C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.
ATP结合盒（ABC）转运蛋白属于一个庞大的蛋白质超家族，它们具有共同的功能和共同的核苷酸结合结构域。来自大肠杆菌的CvaB蛋白是细菌ABC输出子亚家族的成员，对于肽抗生素大肠杆菌素V的输出至关重要。在此我们报告，令人惊讶的是，在低温下，CvaB羧基末端核苷酸结合结构域（BCTD）可优先与GTP而非ATP发生交联。这种交联依赖于Mg2+和Mn2+。然而，BCTD在37℃时具有相似的GTP酶和ATP酶活性，具有相同的动力学参数以及对抑制剂的相似反应。此外，CvaB中的一个点突变（D654H）完全消除了大肠杆菌素V的分泌，严重损害了相应BCTD中的GTP酶和ATP酶活性，表明这两种活性来自同一种酶。有趣的是，ATP的水解活性比GTP对低温更为敏感：BCTD在10℃时主要具有GTP水解活性，这与交联结果一致。这些发现提示了一种ABC蛋白介导的、对GTP水解具有特异性的转运新机制。
