Substrate binding-induced conformational transitions in the omega-3 fatty acid transporter MFSD2A.

Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na-dependent uptake of ω-3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na. Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a "trap-and-flip" mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.