Mechanisms of Cholesterol Binding to LAT1

The human L-type amino acid transporter 1 (LAT1; SLC7A5), is an amino acid exchanger protein, primarily found in the blood-brain-barrier, placenta, and testis, where it plays a key role in amino acid homeostasis. Cholesterol is an essential lipid that has been highlighted to play a role in regulating the activity of membrane transporters such as LAT1, yet little is known about the molecular mechanisms driving this phenomenon. Here we perform a comprehensive computational analysis to investigate cholesterols role in LAT1 structure and function, focusing on four cholesterol binding sites (CHOL1-4) identified in a recent LAT1-apo inward-open conformation cryo-EM structure. We performed four independent molecular dynamics (MD) simulations of LAT1 bound to each cholesterol molecule, as well as molecular docking, free energy calculation by MM/GBSA, and other analysis tools, to investigate LAT1-cholesterol interactions. Our results indicate that CHOL3 provides the most stable binding interactions with LAT1, and CHOL3 and CHOL1 sites have the largest stabilizing effect on LAT1s primary functional motifs (hash and bundle) and substrate binding site. Our analysis also uncovers an alternative cholesterol binding site to the originally assigned CHOL1. Our study improves the understanding of cholesterols modulatory effect on LAT1 and proposes candidate sites for discovery of future allosteric ligands with rational design.