Palmitoylation Depth Determines Lipid-Phase Preference in Claudins

Abstract S-palmitoylation is a reversible lipid modification that regulates membrane protein organization and function, but its molecular consequences remain incompletely understood. Claudins, the primary functional proteins of tight junctions, contain conserved palmitoylation sites on the cytosolic leaflet that influence tight junction assembly and barrier function. Using MARTINI coarse-grained molecular dynamics simulations, we investigated how palmitoylation affects claudin-lipid interactions and membrane phase partitioning in four claudins (CLD3, CLD4, CLD9, and CLD14) in phase-separated bilayers. Palmitoylation enhances claudins’ affinity for ordered phases, promotes local cholesterol enrichment, and reduces disordered-lipid contacts. We find that the membrane-embedded depth of the palmitoyl chain is a key determinant of this preference: sites closer to the membrane interface stabilize claudins within ordered domains, while deeper sites favor disordered environments. This depth dependence arises from differential palmitoyl chain tilting, with upright packing at shallow sites and forced lateral tilting at deeper sites. These results identify palmitoylation depth as a structural determinant of claudin-lipid interactions and provide insight into how this reversible modification governs tight junction protein localization within heterogeneous membranes.