Membrane Curvature Generation by the Caveolin 8S Complex and the Role of Cholesterol

The protein caveolin-1 (Cav1) is essential in the generation of caveolae, but the mechanism of its action remains unclear. A recent cryo-EM structure revealed an 11-mer of Cav1 (the 8S complex) forming a disk with a flat membrane-facing surface, raising the question of how a flat complex is able to generate positive membrane curvature. Previous implicit-solvent and all-atom molecular dynamics simulations showed the 8S complex adopting a conical shape. Here, we simulated the complex in discontinuous membrane patches on the Anton 3 supercomputer. In a 2-μs simulation, a flat POPC membrane patch converted into a hemisphere with the 8S complex on its apex. When the complex was constrained to remain flat, the membrane patch acquired slightly negative curvature. Thus, the conical shape of the 8S complex is essential for positive curvature generation, supporting a scaffolding mechanism of membrane curvature generation by Cav1. The two recently discovered invertebrate caveolins remained flat and generated pronounced negative curvature due to interactions of lipids with the protein rim. Simulations of Cav1 in 70:30 POPC:cholesterol and other cholesterol-containing mixtures showed significantly lower curvature than in the pure POPC membrane or in an E. coli membrane mimic. This appears to be caused by cholesterol moving from the distal to the proximal leaflet. No specific binding of cholesterol to the Cav1 CRAC motif was observed, nor was significant enrichment of cholesterol in contact with the complex. These observations lead to the hypothesis that cholesterol is enriched in caveolae not because of specific binding to caveolin, but because it can alleviate curvature stress due to its negative spontaneous curvature and its ability to rapidly flip-flop.