The mycobacterium lipid transporter MmpL3 is dimeric in detergent solution, SMALPs and reconstituted nanodiscs†

IF 4.2 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Sara Cioccolo, Joseph D. Barritt, Naomi Pollock, Zoe Hall, Julia Babuta, Pooja Sridhar, Alicia Just, Nina Morgner, Tim Dafforn, Ian Gould and Bernadette Byrne
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Abstract

The mycobacterial membrane protein large 3 (MmpL3) transports key precursor lipids to the outer membrane of Mycobacterium species. Multiple structures of MmpL3 from both M. tuberculosis and M. smegmatis in various conformational states indicate that the protein is both structurally and functionally monomeric. However, most other resistance, nodulation and cell division (RND) transporters structurally characterised to date are either dimeric or trimeric. Here we present an in depth biophysical and computational analysis revealing that MmpL3 from M. smegmatis exists as a dimer in a variety of membrane mimetic systems (SMALPs, detergent-based solution and nanodiscs). Sucrose gradient separation of MmpL3 populations from M. smegmatis, reconstituted into nanodiscs, identified monomeric and dimeric populations of the protein using laser induced liquid bead ion desorption (LILBID), a native mass spectrometry technique. Preliminary cryo-EM analysis confirmed that MmpL3 forms physiological dimers. Untargeted lipidomics experiments on membrane protein co-purified lipids revealed PE and PG lipid classes were predominant. Molecular dynamics (MD) simulations, in the presence of physiologically-relevant lipid compositions revealed the likely dimer interface.

Abstract Image

分枝杆菌脂质转运体 MmpL3 在洗涤剂溶液、SMALPs 和重组纳米盘中是二聚体
分枝杆菌膜蛋白大 3(MmpL3)将关键的前体脂质转运到分枝杆菌的外膜。结核分枝杆菌和烟曲霉分枝杆菌的 MmpL3 在不同构象状态下的多个结构表明,该蛋白在结构上和功能上都是单体。然而,迄今为止,大多数其他抗性、结核和细胞分裂(RND)转运体的结构特征要么是二聚体,要么是三聚体。在此,我们进行了深入的生物物理和计算分析,发现在各种膜模拟系统(SMALPs、去垢剂溶液和纳米盘)中,来自烟草真菌的 MmpL3 是以二聚体形式存在的。利用激光诱导液珠离子解吸(LILBID)--一种原生质谱技术--对重组到纳米盘中的来自烟曲霉的 MmpL3 群体进行蔗糖梯度分离,确定了该蛋白质的单体和二聚体群体。初步的冷冻电镜分析证实,MmpL3 形成了生理二聚体。对膜蛋白共纯化脂质进行的非靶向脂质组学实验显示,PE 和 PG 类脂质占主导地位。分子动力学模拟显示,在存在生理相关脂质成分的情况下,可能存在二聚体界面。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.10
自引率
0.00%
发文量
128
审稿时长
10 weeks
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