Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden.
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden; Science for Life Laboratory, Stockholm University, SE-17177 Solna, Sweden.
J Mol Biol. 2014 May 29;426(11):2246-54. doi: 10.1016/j.jmb.2014.03.012. Epub 2014 Mar 30.
The increasing number of solved membrane protein structures has led to the recognition of a common feature in a large fraction of the small-molecule transporters: inverted repeat structures, formed by two fused homologous membrane domains with opposite orientation in the membrane. An evolutionary pathway in which the ancestral state is a single gene encoding a dual-topology membrane protein capable of forming antiparallel homodimers has been posited. A gene duplication event enables the evolution of two oppositely orientated proteins that form antiparallel heterodimers. Finally, fusion of the two genes generates an internally duplicated transporter with two oppositely orientated membrane domains. Strikingly, however, in the small multidrug resistance (SMR) family of transporters, no fused, internally duplicated proteins have been found to date. Here, we have analyzed fused versions of the dual-topology transporter EmrE, a member of the SMR family, by blue-native PAGE and in vivo activity measurements. We find that fused constructs give rise to both intramolecular inverted repeat structures and competing intermolecular dimers of varying activity. The formation of several intramolecularly and intermolecularly paired species indicates that a gene fusion event may lower the overall amount of active protein, possibly explaining the apparent absence of fused SMR proteins in nature.
倒重复结构，由两个融合的同源膜结构域形成，在膜中的方向相反。已经提出了一种进化途径，其中祖先状态是编码能够形成反平行同源二聚体的双拓扑膜蛋白的单个基因。基因复制事件使得能够进化出两种方向相反的形成反平行异源二聚体的蛋白质。最后，两个基因的融合产生了具有两个相反取向的膜结构域的内部复制转运体。然而，令人惊讶的是，在小的多药耐药（SMR）转运蛋白家族中，迄今为止尚未发现融合的、内部复制的蛋白质。在这里，我们通过蓝色非变性 PAGE 和体内活性测量分析了双拓扑转运蛋白 EmrE 的融合版本，该蛋白是 SMR 家族的成员。我们发现融合的构建体既产生了分子内倒重复结构，也产生了具有不同活性的竞争的分子间二聚体。几种分子内和分子间配对种的形成表明基因融合事件可能降低了活性蛋白的总量，这可能解释了自然界中明显缺乏融合的 SMR 蛋白的原因。
