Department of Chemistry, Texas A&M University, College Station, TX, USA.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA.
Nat Commun. 2024 Jul 15;15(1):5946. doi: 10.1038/s41467-024-50350-9.
The ATP-binding cassette (ABC) transporter, MsbA, plays a pivotal role in lipopolysaccharide (LPS) biogenesis by facilitating the transport of the LPS precursor lipooligosaccharide (LOS) from the cytoplasmic to the periplasmic leaflet of the inner membrane. Despite multiple studies shedding light on MsbA, the role of lipids in modulating MsbA-nucleotide interactions remains poorly understood. Here we use native mass spectrometry (MS) to investigate and resolve nucleotide and lipid binding to MsbA, demonstrating that the transporter has a higher affinity for adenosine 5'-diphosphate (ADP). Moreover, native MS shows the LPS-precursor 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-lipid A (KDL) can tune the selectivity of MsbA for adenosine 5'-triphosphate (ATP) over ADP. Guided by these studies, four open, inward-facing structures of MsbA are determined that vary in their openness. We also report a 2.7 Å-resolution structure of MsbA in an open, outward-facing conformation that is not only bound to KDL at the exterior site, but with the nucleotide binding domains (NBDs) adopting a distinct nucleotide-free structure. The results obtained from this study offer valuable insight and snapshots of MsbA during the transport cycle.
ATP 结合盒（ABC）转运蛋白 MsbA 在脂多糖（LPS）生物合成中起着关键作用，通过促进 LPS 前体脂寡糖（LOS）从细胞质转运到内膜的周质小叶。尽管有多项研究阐明了 MsbA 的作用，但脂质在调节 MsbA-核苷酸相互作用中的作用仍知之甚少。在这里，我们使用天然质谱（MS）来研究和解析 MsbA 与核苷酸和脂质的结合，证明该转运蛋白对腺苷 5'-二磷酸（ADP）具有更高的亲和力。此外，天然 MS 表明 LPS 前体 3-脱氧-D-甘露辛-2-酮酸（Kdo）-脂质 A（KDL）可以调节 MsbA 对三磷酸腺苷（ATP）与 ADP 的选择性。受这些研究的启发，确定了四个开放的、内向的 MsbA 结构，它们在开放性上有所不同。我们还报道了一个 2.7 Å 分辨率的 MsbA 开放外向构象结构，它不仅在外部位点与 KDL 结合，而且核苷酸结合域（NBD）采用了独特的无核苷酸结构。这项研究的结果提供了在运输循环中 MsbA 的有价值的见解和快照。
