Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):11367-72. doi: 10.1073/pnas.1302515110. Epub 2013 Jun 24.
Ca(2+) efflux by Ca(2+) cation antiporter (CaCA) proteins is important for maintenance of Ca(2+) homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na(+)/Ca(2+) exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca(2+) efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca(2+)/H(+) antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca(2+)-binding site. A hydrophobic "seal" closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca(2+)/H(+) exchange pathway. In the Ca(2+)-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na(+)-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca(2+)/H(+) exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.
Ca(2+) 通过 Ca(2+) 阳离子反向转运蛋白 (CaCA) 从细胞膜中流出对 Ca(2+) 离子的内稳态平衡很重要。最近，来自 Methanococcus jannaschii 的原核 Na(+)/Ca(2+) 交换器 (NCX) 反向转运蛋白 NCX_Mj 的单体结构显示出向外开放的构象，这表明 Ca(2+) 流出的底物进入是交替的假说。为了证明 CaCA 机制所必需的构象变化，我们呈现了来自 Bacillus subtilis 的 Ca(2+)/H(+) 反向转运蛋白 YfkE 的晶体结构，分辨率为 3.1 Å。YfkE 形成同源三聚体，通过二硫键交联得到证实。质子化状态的 YfkE 表现出向内开放的构象，每个单体的细胞质中都有一个大的亲水腔，在膜中间的 Ca(2+) 结合位点处结束。一个疏水性“密封”关闭其周质出口。四个保守的 α-重复螺旋以 X 样构象组装，形成 Ca(2+)/H(+) 交换途径。在 Ca(2+) 结合位点，两个必需的谷氨酸残基与 NCX_Mj 中的对应残基相比呈现出不同的构象，而几个氨基酸取代则封闭了 Na(+) 结合位点。向内开放的 YfkE 和向外开放的 NCX_Mj 之间的结构差异表明，构象转变是由跨膜螺旋 2 和 7 的拐点角度的旋转触发的，并由其相邻跨膜螺旋 1 和 6 的大构象变化介导。我们的结构和突变分析不仅为 Ca(2+)/H(+) 交换及其 pH 调节的机制奠定了结构基础，而且还揭示了 CaCA 蛋白家族对不同能量模式的进化适应。
