National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing100101, China.
Lingnan Guangdong Laboratory of Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China.
J Mol Biol. 2021 Jun 25;433(13):166986. doi: 10.1016/j.jmb.2021.166986. Epub 2021 May 11.
The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4-3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaFEBD, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.
革兰氏阴性菌的外膜（OM）由外叶的脂多糖（LPS）和内叶的磷脂（PL）组成，在外膜抗生素耐药性和病原体毒力中起着关键作用。已知脂质不对称性（Mla）途径参与 PL 转运，并有助于 OM 的脂质动态平衡，但该途径中 PL 转运的潜在分子机制和方向仍不清楚。在这里，我们报道了铜绿假单胞菌 MlaFEBD 的 cryo-EM 结构，这是 Mla 途径的核心复合物，处于无核苷酸（apo）、ADP（ATP+钒酸盐）和 ATP（AMPPNP）结合状态，以及大肠杆菌 MlaFEB 的结构，处于 apo 和 AMPPNP 结合状态，分辨率范围为 3.4-3.9Å。这些结构表明，MlaFEBD 复合物总共包含 12 个蛋白质分子，其化学计量比为 MlaFEBD，并在不同位置结合大量 PL。与典型的 ABC 转运体不同，核苷酸结合并不能触发 ABC 转运体的 MlaFEBD 和 MlaFEB 在核苷酸结合和跨膜结构域中的显著构象变化，这与它们在去污剂胶束和脂质纳米盘中表现出的低 ATP 酶活性有关。有趣的是，在添加 ATP 后，PL 或去污剂似乎从由 MlaD 六聚体形成的疏水隧道的远端迁移到膜近端，表明 PL 可能以 ATP 依赖的方式在隧道中逆行转运。定点光交联实验证实，二聚体 MlaE 和 MlaD 六聚体的底物结合口袋能够在体外结合 PL，这与 MlaFEBD 复合物作为 PL 转运体的概念一致。
