Alternating access of the putative substrate-binding chamber in the ABC transporter MsbA.
Alternating access of the putative substrate-binding chamber in the ABC transporter MsbA.
复制标题
交替进入 ABC 转运蛋白 MsbA 中假定的底物结合室。
DOI：
10.1016/j.jmb.2009.08.051
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发表时间：
2009
影响因子：
5.6
通讯作者：
McHaourab,HassaneS
中科院分区：
文献类型：
--
作者：
Zou,Ping;McHaourab,HassaneS
MsbA是来自大肠杆菌的ATP结合盒转运蛋白，参与脂质A穿过内膜的运输。ATP结合盒转运蛋白利用ATP结合和水解的自由能来驱动底物逆着其浓度梯度向上移位。蛋白质运动耦合能量输入做功的模型受到MsbA晶体学快照的启发。该模型的核心是跨膜室的可及性的切换，涉及底物结合，从向内到向外的配置。在这里，我们使用自旋标记和电子顺磁共振光谱系统地探讨在ATP水解周期中的MsbA结构的重排。自旋标记的可及性和局部动力学确定在脂质体中的无核苷酸中间体和ATP水解的过渡态。这两种中间体之间的电子顺磁共振参数的变化符合与交替进入腔室一致的全局模式。在ATP水解的过渡态，自旋标签的细胞质侧报告增加动态限制和减少水的可及性，而那些在细胞外侧报告增加水的渗透。此外，自旋标记迁移率和可及性以及它们的变化与基于晶体结构的预期一致。室水合作用的逆转可能降低两亲性底物结合的自由能并促进跨膜易位。
MsbA is an ATP-binding cassette transporter from Escherichia coli that is involved in trafficking lipid A across the inner membrane. ATP-binding cassette transporters harness the free energy of ATP binding and hydrolysis to drive the uphill translocation of substrates against their concentration gradients. A model of protein motion coupling energy input to work was inspired by crystallographic snapshots of MsbA. Central to this model is a switch in the accessibility of a transmembrane chamber, implicated in substrate binding, from an inward- to an outward-facing configuration. Here, we used spin labeling and electron paramagnetic resonance spectroscopy to systematically explore rearrangements in MsbA structure during the ATP hydrolysis cycle. Spin-label accessibility and local dynamics were determined in liposomes for the nucleotide-free intermediate and the transition state of ATP hydrolysis. The changes in the electron paramagnetic resonance parameters between these two intermediates fit a global pattern consistent with alternating access of the chamber. In the transition state of ATP hydrolysis, spin labels on the cytoplasmic side report increased dynamic restrictions and reduced water accessibility, while those on the extracellular side report increased water penetration. Furthermore, spin-label mobility and accessibility as well as their changes are consistent with those expected based on the crystal structures. The reversal in chamber hydration is likely to reduce the free energy of amphipathic substrate binding and promote translocation across the membrane.