Elucidating the mechanism of lipid exchange by cholesteryl ester transfer protein (CETP)

Abstract

Cholesteryl Ester Transfer Protein (CETP) exchanges cholesteryl esters (CEs) and triglycerides (TGs) between lipoproteins, modulating their composition. This process regulates plasma CE and TG content and therefore strongly correlates with cardiovascular disease (CVD) risk factors. While CETP is a significant therapeutic target, its mechanism of lipid transfer, particularly the role of its structural components, such as its cavity and associated phospholipids, remains incompletely understood. Identification of molecular determinants that can influence CETP activity is paramount for designing and evaluating next-generation therapeutics. Therefore, in this work, we have employed advanced molecular dynamics simulations to track the movement of lipids through CETP, highlighting the regulatory interactions that influence their lipid transfer efficiency and specificity. CETP encloses a tunnel that serves as a medium for lipid exchange; the movement of lipids can only be understood post identification and characterization of this tunnel. Although several methods exist for protein cavity identification, they are generally limited by the type of cavity they can identify, as well as the automation and resolution of their cavity detection. We addressed these limitations of existing methods by developing CICLOP (Characterization of Inner Cavity Lining of Proteins), a new method utilizing a hybrid grid- and tessellation-based approach to identify internal cavities (tunnels, channels, pores, and voids). CICLOP offers superior performance and automation, accurately identifying the protein cavity and its functional characterization (diameter, volume, hydrophobicity, charge, and conservation). Applying CICLOP, we identified a single, continuous hydrophobic tunnel within CETP that contradicted earlier models that hypothesized diffused, smaller cavities within CETP. Next, we determined the lipid transfer mechanism using steered MD simulations. The CETP cavity, in addition to its terminal openings, has two additional openings, which are plugged by two phospholipids (PLs); however, the role of these openings in CETP structure and function has remained elusive to date. Our structural and functional analyses revealed that lipid traversal through CETP’s central tunnel is facilitated through hydrophobic interaction-mediated diffusion. Moreover, the tunnel’s function is dictated by its dynamic plasticity that allows lipid movement through a peristaltic wave-like motion. Further, the PLs are indispensable in establishing the optimal architecture of the CETP tunnel and accelerating lipid traversal through a novel “gliding” mechanism. Using free energy calculations and in vitro mutagenesis, an in-depth understanding of the mechanism of lipid exchange by CETP, guided and accentuated by its interaction with PLs, was obtained. To further understand the complete lipid transfer mechanism, we investigated both the global process and lipid-specific factors governing movement. Our free energy calculations demonstrated the non-specificity of the CETP termini for lipid entry, indicating that this process is primarily governed by the lipid’s surface availability, rather than any inherent protein bias for CE or TG. Furthermore, both lipid types follow a single, con- served physical path once inside the tunnel. Importantly, the CETP tunnel can accommodate two lipids simultaneously moving in opposite directions, providing compelling. The dynamics of lipid traversal are significantly influenced by lipid-specific factors such as acyl chain length and conformation. Long-chain TGs (LCTs) in specific conformations like ’Fork’ and ’T’ exhibit the longest residence times because they form a greater number of stable hydrophobic contacts with the tunnel residues. This finding is critical in the context of cardiovascular disease (CVD) where the slower transfer kinetics of LCTs—which are prevalent in the plasma of CVD patients— may lead to adverse, pro-atherogenic lipoprotein remodeling outcomes. Altogether, this study advances our understanding of CETP by revealing that its function relies on a precisely orchestrated interplay of tunnel plasticity and optimal hydrophobicity, which allows lipid entry and diffusion through the tunnel. The study also identified PL-plugs as essential co-factors that strongly influenced CETP function, with their ability to modulate both the tunnel hydrophobicity and lipid transfer dynamics. Lipid-specific factors, such as acyl chain length, which are directly influenced by diet, also influence lipid transfer dynamics. The study provides compelling evidence in favor of a ternary complex, wherein CETP bridges two lipoproteins, simultaneously facilitating the concurrent exchange of CE for TG. These insights open new avenues for designing and evaluating next-generation CETP-targeted drugs by focusing on the solvent-accessible PL-binding pockets or the integrity of the tunnel, providing a refined approach for modulating CETP function in patients with atherosclerosis.