Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae
Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae
复制标题
DOI：
10.1073/pnas.1802417115
复制
发表时间：
2018-07-03
影响因子：
11.1
通讯作者：
Mchaourab, Hassane S.
中科院分区：
文献类型：
--
作者：
Claxton, Derek P.;Jagessar, Kevin L.;Mchaourab, Hassane S.
属于多药和有毒化合物挤出（MATE）家族的次级活性转运蛋白利用电化学离子梯度的势能输出广谱的细胞毒性化合物，从而有助于多药耐药性。目前对离子耦合底物传输机制的理解是通过来自多种生物体的一组有限的 MATE 转运蛋白晶体结构来了解的，这些结构捕获了采用类似的外向构象的 12 跨膜螺旋拓扑。尽管这些结构映射了对功能重要的保守残基，但这些残基在形成构象循环中的机制作用尚未得到研究。在这里，我们使用双电子电子共振 (DEER) 光谱来探索霍乱弧菌 (NorM-Vc) 中 NorM 的配体依赖性构象变化，NorM-Vc 是一种与 Na+ 和 H+ 梯度耦合的 MATE 转运蛋白。 NorM-Vc 周质侧自旋标记之间的距离测量确定了由 Na+、H+ 或底物阿霉素结合诱导的独特结构中间体。 Na+ 和 H+ 依赖性中间体与 TM1 的不同构象相关。保守残基的定点诱变表明，N 端结构域空腔中的极性残基网络促进了 Na+ 和 H+ 驱动的构象变化，而埋藏在 C 端结构域中的保守羧酸盐对于稳定药物结合状态至关重要。结合突变型 NorM-Vc 的阿霉素结合和细胞毒性测定进行解释，这些结果确立了离子耦合构象动力学在功能循环中的作用，并暗示 H+ 在阿霉素释放机制中的作用。
Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from Vibrio cholerae (NorM-Vc), a MATE transporter proposed to be coupled to both Na+ and H+ gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na+, H+, or the substrate doxorubicin. The Na+-and H+-dependent intermediates were associated with distinct conformations of TM1. Sitedirected mutagenesis of conserved residues revealed that Na+-and H+-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H+ in the doxorubicin release mechanism.