Phospholipids that plug the pores of cholesteryl ester transfer protein control its lipid transfer dynamics

The mechanism by which cholesteryl ester transfer protein (CETP), a key drug target in cardiovascular disease, moves lipids between lipoproteins, has remained elusive. CETP is the primary protein that regulates plasma circulating levels of lipids (within high, low, and very low-density lipoproteins. The protein, in addition to its terminal openings, has two additional openings, plugged by two phospholipids (PLs). Small molecule inhibitors targeting CETP tunnel displace PL during CETP inhibition. Here, using steered molecular dynamics simulations followed by in-vitro mutagenesis we show that CETP-bound PLs are indispensable in establishing the optimal architecture of CETP tunnel. Our structural and functional analyses revealed that lipid traversal through CETPs central tunnel is facilitated through hydrophobic interaction-mediated diffusion. PLs are critical in synchronizing domain movements of CETP while accelerating triglyceride traversal through the tunnel and their activity regulated through salt-bridge interactions. Most notably, we show for the first time that phospholipids (PLs) bound within CETPs tunnel accelerate lipid movement through a novel "gliding" mechanism, representing the first known example of a lipid regulating the movement of another. We identified conserved phenylalanine (Phe) flaps that act as a regulatory valve, opening and closing concertedly to prevent lipid backflow in the absence of an active motor. This study provides in-depth understanding of the mechanism of lipid exchange by CETP guided, and accentuated by its interaction with PLs. The conservation of these critical structural elements across the BPI/LBP family suggests that this mechanism is broadly applicable, expanding our understanding of lipid transport in this clinically significant protein class. Significance StatementThe mechanism of lipid transport by the cholesteryl ester transfer protein (CETP), a major drug target, has long been a scientific mystery. Neutral lipid exchange through CETP is dependent on the hydrophobic characteristics of the tunnel that has been exploited during inhibitor design. We provide a detailed, molecular-level explanation, showing for the first time that phospholipids (PLs), previously thought to be static plugs, are indispensable for lipid transfer. They actively accelerate lipid movement through a novel gliding mechanism, the first documented instance of a lipid regulating another. This discovery identifies new therapeutic targets for CETP inhibition. Our findings also suggest that this fundamental mechanism is conserved across a large protein family, with broad implications for understanding lipid transport and function.