The NPC1L1 Sterol Transporter: A Molecular Dynamics Study on the Influence of Cholesterol Binding on Intradomain and Interdomain Motions

Abstract The Niemann-Pick C1-Like 1 (NPC1L1) transmembrane protein, localized on the apical membrane of enterocytes, is a key player in cholesterol metabolism and homeostasis, whose proper regulation is essential to avoid the development of cardiovascular diseases. The cryo-EM resolution of its structure in two distinct conformations provided a possible explanation of the mechanism which allows cholesterol to be transported from the lumen to the cytoplasm. This mechanism likely exploits the high mobility of the luminal N-terminal domain (NTD), which first binds the cholesterol molecule─although this was not resolved in the cryo-EM structures─ and whose spatial disposition with respect to the other extracellular domains is what most distinguishes the two conformations. In this work, we performed extensive all-atom molecular dynamics (MD) simulations of the NPC1L1 protein, embedded in a membrane model, in four functional states: open apo, open bound, closed bound, and closed apo. Briefly, the analyses of the MD trajectories showed that the wide motions of NTD observed in the open apo state are partially restrained in the open bound state. Moreover, cholesterol binding to the NTD also makes this domain dynamically coupled to the collective rearrangements of the middle luminal domain (MLD) and the C-terminal domain (CTD). In the closed bound state, a new NTD-cholesterol hydrogen bond is observed, which could represent the first step for cholesterol delivery. Finally, the closed apo state is characterized by an increased MLD flexibility which may destabilize extracellular domain interfaces, preparing the NPC1L1 transporter to return to the open apo state. Ultimately, our protocol provides a robust framework that can be used to analyze novel NPC1L1 inhibitors.