Conformational cycle of the ABC transporter MsbA in liposomes: detailed analysis using double electron-electron resonance spectroscopy.
Conformational cycle of the ABC transporter MsbA in liposomes: detailed analysis using double electron-electron resonance spectroscopy.
复制标题
DOI：
10.1016/j.jmb.2009.08.050
复制
发表时间：
2009-10-30
影响因子：
5.6
通讯作者：
Mchaourab, Hassane S.
中科院分区：
文献类型：
--
作者：
Zou, Ping;Bortolus, Marco;Mchaourab, Hassane S.
关键词：
在ATP结合和水解的能量驱动下，ATP结合盒（ABC）转运蛋白在向内和向外构象之间交替，允许底物的矢量运动。已经提出了一种新的构象运动模型来描述这种转换在运输途径中的作用。一个模型，基于三个晶体结构的脂质翻转酶MsbA，设想一个大幅度的运动，脱离核苷酸结合域和重新包装的跨膜螺旋。为了测试这个模型，并放置在一个机械的上下文中的晶体结构，我们使用自旋标记和双电子电子共振（DEER）光谱来定义的性质和幅度MsbA的构象变化在ATP水解周期。为此目的，自旋标签被引入到选定的网站，以提供独特的模式的距离变化独特的结晶转变。脂质体中的距离变化，由无核苷酸MsbA到最高能量中间体的转变引起，符合一个简单的模式，其中细胞质侧的残基进行20- 30 π的闭合运动，而在细胞外侧观察到7- 10 π的打开运动。跨膜螺旋经历相对运动以产生与晶体结构所暗示的一致的向外开口。DEER距离分布揭示了与底物结合室的不对称开放相关的转运蛋白两侧的不对称骨架柔性。再加上广泛的可及性分析，我们的研究结果表明，这些结构捕捉功能的运动，耦合ATP能量消耗的工作提供了一个框架的机制，基板运输。
Driven by the energy of ATP binding and hydrolysis, ATP binding cassette (ABC) transporters alternate between inward- and outward-facing conformations allowing vectorial movement of substrates. Conflicting models have been proposed to describe the conformational motion underlying this switch in access of the transport pathway. One model, based on three crystal structures of the lipid flippase MsbA, envisions a large amplitude motion that disengages the nucleotide binding domains and repacks the transmembrane helices. To test this model and place the crystal structures in a mechanistic context, we use spin labeling and Double Electron Electron Resonance (DEER) spectroscopy to define the nature and amplitude of MsbA conformational change during ATP hydrolysis cycle. For this purpose, spin labels were introduced at sites selected to provide a distinctive pattern of distance changes unique to the crystallographic transformation. Distance changes in liposomes, induced by the transition from nucleotide-free MsbA to the highest energy intermediate, fit a simple pattern whereby residues on the cytoplasmic side undergo 20–30Å closing motion while a 7–10Å opening motion is observed on the extracellular side. The transmembrane helices undergo relative movement to create the outward opening consistent with that implied by the crystal structures. DEER distance distributions reveal asymmetric backbone flexibility on the two sides of the transporter that correlates with asymmetric opening of the substrate binding chamber. Together with extensive accessibility analysis, our results suggest that these structures capture features of the motion that couples ATP energy expenditure to work providing a framework for the mechanism of substrate transport.
登录
查看更多内容
影响因子：
6.8
作者：
Oldham, Michael L.;Davidson, Amy L.;Chen, Jue
通讯作者：
Chen, Jue
影响因子：
56.9
作者：
Locher, KP;Lee, AT;Rees, DC
通讯作者：
Rees, DC
影响因子：
64.8
作者：
Oldham, Michael L.;Khare, Dheeraj;Chen, Jue
通讯作者：
Chen, Jue
影响因子：
15
作者：
Borbat, PP;Mchaourab, HS;Freed, JH
通讯作者：
Freed, JH
影响因子：
2.2
作者：
Chiang, YW;Borbat, PP;Freed, JH
通讯作者：
Freed, JH