Beyond the Crystal: Molecular Dynamics Investigations of CETP with Varied Lipid Substrates Reveal Asymmetric Dominant Motions

Cholesteryl ester transfer protein (CETP) is a crucial therapeutic target for combating cardiovascular disease (CVD) due to its strong influence in modulating high-density lipoprotein (HDL) levels. CETP is responsible for the bidirectional transfer of cholesteryl esters (CEs) and triglycerides (TGs) between different lipoprotein fractions. Although CETP encounters both these neutral lipid substrates when it penetrates deep into lipoprotein cores and can acquire either lipid, prior studies have examined its conformational space only in the presence of CEs or TGs individually. Here, we investigate the uncharacterised dynamics of CETP in heterogeneous lipid environments (CE-TG and TG-CE) using molecular dynamics simulations. Compared to the stable, homogeneous CE-bound state, the introduction of TG, particularly in mixed CE/TG configurations, induces significant structural instability and protein expansion. Mixed-lipid occupancy leads to elevated flexibility in critical lipoprotein-binding loops and the distortion of vital secondary structural elements. Furthermore, large-scale collective motion analyses reveal that heterogeneous binding forces CETP into aberrant, asymmetric, and hyper-twisted conformations. This disrupts the symmetric bending-twisting balance essential for efficient lipid exchange. Free energy landscapes confirm that the TGs within the mixed-lipid systems exhibit varied conformational states and adopt orientations that deviate from their reported parallel N-N orientation for lipid transfer through CETP. These findings suggest that the simultaneous presence of CE and TG imposes considerable conformational strain, fundamentally impairing CETP's lipid transport mechanism and offering novel mechanistic insights for future CETP-targeted therapeutics.