Specific cholesterol binding drives drastic structural alterations in Apolipoprotein A1

Abstract

Protein adopts multitude of flexible and rapidly interconverting conformers, many which are governed by specific protein-interaction domains. ApoA1, a key player involved in high-density lipoprotein (HDL) regulation exists in structurally diverse forms with varying degree of cholesterol association, and each state is associated with different functional properties. While disc-shaped HDL and its oligomeric ApoA1 protein components have been the focus of several investigations, structural properties of monomeric ApoA1 are poorly understood. Here, we undertook large-scale structural analysis of ApoA1 in apo and cholesterol-bound forms using tens of independent simulations with total computing time exceeding 50 s. Examination of multiple lipid-free trajectories of monomeric ApoA1 revealed a common conformation, with distinct spatial proximity between N- and C-terminal domains. With incorporation of physiologically known cholesterol concentration ({approx}100 cholesterol molecules) in ApoA1 simulations, the monomeric protein spontaneously formed an open circular topology. Remarkably, these drastic structural perturbations are driven by specific binding site at C-terminal and a novel cholesterol binding site at the N-terminal. We proposed a mechanism of stage-wise opening of ApoA1 and demonstrated that less cholesterol concentration around interaction sites or mutation within N-terminal binding sites does not lead to open bell-shaped topology. The kinetic barriers between open and closed-states also showed an ensemble of loosely packed helix bundle (H1-H7; H4-H7) that posed as a slow-intermediate step. Lastly we performed complementary experiments, including ITC and CD measurements to confirm that structural changes are induced by ligand association and not driven by random hydrophobic effect. Collectively, our study suggests a previously unknown mechanism of cholesterol sequestering by ApoA1 that could directly aid in developing modulators for cholesterol efflux with chronic cardiovascular diseases.