The extracellular gate shapes the energy profile of an ABC exporter
The extracellular gate shapes the energy profile of an ABC exporter
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DOI：
10.1038/s41467-019-09892-6
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发表时间：
2019-05-21
影响因子：
16.6
通讯作者：
Seeger, Markus A.
中科院分区：
文献类型：
--
作者：
Hutter, Cedric A. J.;Timachi, M. Hadi;Seeger, Markus A.
ABC输出者利用ATP的能量将底物泵过膜。细胞外闸门的打开和关闭是转运循环的关键步骤，但其基本机制知之甚少。在这里，我们产生了一种合成的单域抗体（sybody），其仅在ATP存在下识别异源二聚体ABC输出蛋白TM 287/288，这对于解决外向转运蛋白的3.2埃晶体结构是必不可少的。该系统结合到细胞外翼，并通过将转运蛋白的构象平衡向外向状态移动来强烈抑制ATP酶活性，如双电子-电子共振（DEER）所示。促进细胞外门开放的突变导致相当的平衡偏移，并强烈降低ATP酶活性和药物转运。使用的sybody作为构象探针，我们证明，有效的细胞外的门关闭需要解离的NBD二聚体ATP水解后重置转运回到其面向内的状态。
ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 angstrom crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.