title: Structural Insights into the Transport Mechanism of the Human Sodium-dependent Lysophosphatidylcholine Transporter MFSD2A.
abstract: Major facilitator superfamily domain containing 2A (MFSD2A) was recently characterized as a sodium-dependent lysophosphatidylcholine transporter expressed at the blood-brain barrier endothelium. It is the primary route for importation of docosohexaenoic acid and other long-chain fatty acids into fetal and adult brain and is essential for mouse and human brain growth and function. Remarkably, MFSD2A is the first identified major facilitator superfamily member that uniquely transports lipids, implying that MFSD2A harbors unique structural features and transport mechanism. Here, we present three three-dimensional structural models of human MFSD2A derived by homology modeling using MelB- and LacY-based crystal structures and refined by biochemical analysis. All models revealed 12 transmembrane helices and connecting loops and represented the partially outward-open, outward-partially occluded, and inward-open states of the transport cycle. In addition to a conserved sodium-binding site, three unique structural features were identified as follows: a phosphate headgroup binding site, a hydrophobic cleft to accommodate a hydrophobic hydrocarbon tail, and three sets of ionic locks that stabilize the outward-open conformation. Ligand docking studies and biochemical assays identified Lys-436 as a key residue for transport. It is seen forming a salt bridge with the negative charge on the phosphate headgroup. Importantly, MFSD2A transported structurally related acylcarnitines but not a lysolipid without a negative charge, demonstrating the necessity of a negatively charged headgroup interaction with Lys-436 for transport. These findings support a novel transport mechanism by which lysophosphatidylcholines are "flipped" within the transporter cavity by pivoting about Lys-436 leading to net transport from the outer to the inner leaflet of the plasma membrane.
authors.affiliation: From the Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Graduate Medical School, Singapore 169857.
authors.forename: Debra Q Y
authors.initials: DQ
authors.lastname: Quek
authors.affiliation: the Department of Biochemistry, Yong Loo Lin School of Medicine, and.
authors.forename: Long N
authors.initials: LN
authors.lastname: Nguyen
authors.affiliation: the Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 138671 Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore 117545, and fanh@bii.a-star.edu.sg.
authors.forename: Hao
authors.initials: H
authors.lastname: Fan
authors.affiliation: From the Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Graduate Medical School, Singapore 169857, david.silver@duke-nus.edu.sg.
authors.forename: David L
authors.initials: DL
authors.lastname: Silver
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008244:Lysophosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D027981:Symporters; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008244:Lysophosphatidylcholines; C549328:MFSD2A protein, human; D027981:Symporters; D025521:Tumor Suppressor Proteins
keywords: X-ray crystallography; blood-brain barrier; brain metabolism; docosahexaenoic acid; drug transport; lysophospholipid; membrane protein; membrane transporter; microcephaly; polyunsaturated fatty acid (PUFA)