Structural Insights into Recognition and Translocation of Oxidized Phospholipid by CD36 Using Mass Spectrometry, Molecular Docking, Dynamics, and Metadynamics Simulations.

CD36 is a multifunctional receptor widely expressed in immune and nonimmune cells, known for its role in lipid transport and inflammatory signaling. Oxidized phospholipids (oxPLs), a class of prominent lipid oxidation products generated under oxidative stress, bind CD36 with high affinity, contributing to the development of atherogenesis and thrombosis and potentially influencing other CD36-dependent biological events. The molecular basis for the oxPL-CD36 interaction is poorly understood. Here, we used cutting-edge enrichment-mass spectrometry to identify lysine residues of CD36 that directly interact with oxPLs. These residues are located along a putative ligand translocation path─spanning from the apex of the extracellular domain to the entrance, interior, and around the exit of the lipid transport tunnel. Molecular docking revealed two sets of oxPL binding poses: one within a tunnel and the other on a surface loop cluster spanning the top to midsection, including the tallest loop containing oxPL-modified K398/K403. These findings support the selective oxPL binding observed in the LC-MS/MS analysis. Molecular dynamics (MD) simulation demonstrated that the sn-1 chain and headgroup of oxPLs engage distinct CD36 residues through hydrophobic, hydrogen-bonding, and ionic interactions, optimally positioning the reactive sn-2 group for lysine modification. MD and metadynamics simulations further demonstrated oxPL translocation through the tunnel, beginning with sn-1 chain insertion, followed by reorientation at the tunnel midsection, where the sn-2 chain and sn-3 headgroup lead the molecule toward the exit. Together, these studies indicate that CD36 may serve as a transporter of individual oxPL molecules into the cell and outline a translocation pathway, key residues and binding forces involved.