McMurry L M, Levy S B
Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Biol Chem. 1995 Sep 29;270(39):22752-7. doi: 10.1074/jbc.270.39.22752.
The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively). An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer. In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives. TetA, TetA alpha, and TetA beta were each fused genetically to a six-histidine carboxyl-terminal tail. The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined. An interaction between alpha domains on different polypeptides which resulted in multimerization was seen. The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein. No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations. A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented.
来自Tn10的43.1 kDa四环素 - 阳离子/质子反向转运蛋白TetA由两个大小相等的结构域组成，即α结构域和β结构域（分别为氨基末端和羧基末端的一半）。α结构域中的失活突变可以补偿β结构域中第二条多肽上的突变，从而在细菌细胞中恢复部分四环素抗性，这表明分子间相互作用使这种转运蛋白能够作为多聚体发挥作用。在本研究中，在含有不同TetA衍生物的大肠杆菌细胞膜的十二烷基麦芽糖苷提取物混合物中检测了多聚体的形成。TetA、TetAα和TetAβ分别通过基因融合到一个六个组氨酸的羧基末端尾巴上。测定了固定在镍亲和柱上的这些融合蛋白与野生型TetA或其他Tet融合蛋白结合的能力。观察到不同多肽上的α结构域之间存在导致多聚化的相互作用。这种结合对Tet蛋白具有特异性，与其他膜蛋白或另一种多组氨酸融合蛋白不发生结合。通过这种方法未检测到α - β相互作用，尽管基于α - β基因互补推测它们在完整细胞中会发生。提出了一个具有分子间α - α和α - β相互作用的TetA二聚体模型。
