Structural basis for lipid and copper regulation of the ABC transporter MsbA

• Published in: Nature communications 2022-11, Vol.13 (1), p.7291-11, Article 7291
• Main Authors: Lyu, Jixing, Liu, Chang, Zhang, Tianqi, Schrecke, Samantha, Elam, Nicklaus P., Packianathan, Charles, Hochberg, Georg K. A., Russell, David, Zhao, Minglei, Laganowsky, Arthur
• Format: Article
• Language: English
• Subjects: 101/28; 101/58; 631/1647/296; 631/326/41/2536; 631/45/612/1237; 631/535/1258/1259; 82/80; 82/83; ABC transporter; ABC transporters; Adenosine; Adenosine Diphosphate - metabolism; Affinity; Allosteric properties; ATP-Binding Cassette Transporters - metabolism; Bacterial Proteins - metabolism; Binding sites; Biosynthesis; Copper; Copper - metabolism; Crystallography; Escherichia coli - metabolism; Humanities and Social Sciences; Lipid A; Lipids; Lipooligosaccharides; Lipopolysaccharides; Lipopolysaccharides - metabolism; Mass spectrometry; Mass spectroscopy; multidisciplinary; Precursors; Science; Science (multidisciplinary); Scientific imaging; Selectivity; Spectroscopy; Structure-function relationships; Ulosonic acid; Vanadate; X-ray crystallography
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-34905-2

A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D- manno -oct-2-ulosonic acid (Kdo) 2 -lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5’-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis. The bacterial ABC transporter MsbA is essential for lipopolysaccharide biogenesis. Here, the authors apply native mass spectrometry, X-ray crystallography, cryo-EM and biochemical approaches to characterize the structural basis and functional roles of MsbA binding to copper and specific lipids.