Structural insights into the transport mechanism of the human sodium-dependent lysophosphatidylcholine transporter Mfsd2a
Debra Q.Y. Quek1, Long N. Nguyen2, Hao Fan3 and David L. Silver1*
- Author Affiliations
1 Duke-NUS Medical School, Singapore;
2 National University of Singapore, Singapore;
3 A*STAR, Singapore
↵* Corresponding author; email: david.silver@duke-nus.edu.sg
Author contributions: D.L.S., D.Q.Q., and H.F. designed research; D.Q.Q. and H.F. performed research; L.N.N performed experiments for Fig. 3H; D.Q.Q. and H.F. performed structural modelling and analysis. D.Q.Q., D.L.S., and H.F. wrote the paper.
Abstract
Major Facilitator Superfamily Domain containing 2A (Mfsd2a) was recently characterized as a sodium-dependent lysophosphatidylcholine (LPC) 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 foetal and adult brain, and is essential for mouse and human brain growth and function. Remarkably, Mfsd2a is the first identified MFS family member that uniquely transports lipids, implying that Mfsd2a harbours unique structural features and transport mechanism. Here, we present three 3D structural models of human Mfsd2a derived by homology modelling 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: 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 Lys436 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 negative charged headgroup interaction with Lys436 for transport. These findings support a novel transport mechanism by which LPCs are flipped within the transporter cavity by pivoting about Lys436 leading to net transport from the outer to the inner leaflet of the plasma membrane.
brain metabolism drug transport lysophospholipid membrane protein polyunsaturated fatty acid (PUFA) X-ray crystallography blood-brain barrier docosahexaenoic acid membrane transporter microcephaly
Received February 9, 2016. Accepted March 4, 2016.
Copyright © 2016, The American Society for Biochemistry and Molecular Biology
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