High Speed AFM Imaging of Structure and Dynamics of Bacterial ABC Transporter MsbA During Lipid Transport
High Speed AFM Imaging of Structure and Dynamics of Bacterial ABC Transporter MsbA During Lipid Transport
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脂质运输过程中细菌 ABC 转运蛋白 MsbA 的结构和动力学的高速 AFM 成像
DOI：
10.1016/j.bpj.2019.11.482
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发表时间：
2020
影响因子：
3.4
通讯作者：
Kien Xuan Ngo
中科院分区：
文献类型：
--
作者：
玉田知之;佐野智紀;長野真;十島純子;十島二朗;Kien Xuan Ngo
从细菌到人类，所有的atp结合盒（ABC）转运体都有一个共同的结构组织，包括两个不同的结构域（即跨膜结构域（TMDs）和胞质核苷酸结合结构域（nbd））。MsbA是一种ABC转运蛋白，通过革兰氏阴性菌的细胞质膜输出脂质A。MsbA还出口药物，赋予多药耐药性。结合不同核苷酸的MsbA的晶体和低温电镜快照结构表明，在两个nbd中，随着atp酶的反应，TMDs可能发生了较大的构象变化。因此，两个nbd必须在多个atp酶周期中重复耦合和解耦。这些动态事件很可能传递到与nbd相连的两个α-螺旋束，从而使tmd （α-螺旋束的主要部分）发生不同的构象，包括内向和外向的构象，从而促进底物的进入、转运和出口。然而，在这些功能活动中，MsbA的结构和动力学并没有同时得到验证。利用HS-AFM的优势，我们成功地实时成像了与不同核苷酸结合的洗涤剂溶解MsbA的结构和动力学，以跟踪它们的构象变化。分析了NBDs和TMDs中开放和封闭构象的动力学，以评估耦合和不耦合NBDs如何改变TMDs中的结构动力学。接下来，在不同核苷酸和药物存在的情况下，实时成像嵌入含有脂质A的脂质膜内的MsbA的tmd通道和nbd的结构动力学。脂质A通过TMDs通道的易位被成功地记录在脂质膜内可控定向的MsbA分子中。综上所述，在脂质A转运过程中，随着NBDs中ATP水解，TMDs的动态构象变化揭示了细菌ABC转运体的一个非常重要的运作机制。
All ATP-binding cassette (ABC) transporters that operate in all species from bacteria to human share a common structural organization, consisting of two distinct domains (ie, transmembrane domains (TMDs) and cytosolic nucleotide binding domains (NBDs)). MsbA, an ABC transporter, exports lipid A across the cytoplasmic membrane of Gram-negative bacteria. MsbA also exports drugs, conferring multidrug resistance. The crystal and cryo-EM snapshot structures of MsbA bound with different nucleotides have suggested that large conformational changes possibly take place in the TMDs accompanied the ATPase reaction in two NBDs. Therefore, the two NBDs must repeat coupling and uncoupling during multiple ATPase cycles. These dynamic events very likely transmit to the two α-helical bundles connected with the NBDs, so that the TMDs (major parts of the α-helical bundles) undergo different conformations including inward-facing and outward-facing conformations that facilitate substrate entry, translocation and export. However, this structure and dynamics of MsbA during these functional activities have not been verified simultaneously. Using HS-AFM advantage, the structures and dynamics of detergent-solubilized MsbA bound with different nucleotides were successfully real-time imaged to follow their conformational changes. The kinetics of open and closed conformations in NBDs and TMDs was analyzed to evaluate how coupling and uncoupling NBDs could change structural dynamics in TMDs. Next, structural dynamics of TMDs channel and NBDs of MsbA embedded within lipid membrane containing lipid A was also real-time imaged in the presence of different nucleotides and drugs. The translocation of lipid A across TMDs channel was successfully recorded in the controllably oriented MsbA molecules within lipid membrane. Taken together, the dynamic conformational changes in TMDs accompanying ATP hydrolysis in NBDs during lipid A transport unveil a very important operational mechanism in bacterial ABC transporters.