Binding of Cholesterol to the N-Terminal Domain of the NPC1L1 Transporter: Analysis of the Epimerization-Related Binding Selectivity and Loop Mutations.

Cholesterol is a fat-like substance with a pivotal physiological relevance in humans, and its homeostasis is tightly regulated by various cellular processes, including the import in the small intestine and the reabsorption in the biliary ducts by the Niemann-Pick C1 Like 1 (NPC1L1) importer. NPC1L1 can mediate the absorption of a variety of sterols but strikingly exhibits a large sensitivity to cholesterol epimerization. This study examines the molecular basis of the epimerization-related selective binding of cholesterol by combining extended unbiased molecular dynamics simulations of the apo and holo species of the N-terminal domain of wild-type NPC1L1, in conjunction with relative binding free energy, umbrella sampling, and well-tempered metadynamics calculations. The analysis of the results discloses the existence of two distinct binding modes for cholesterol and epi-cholesterol. The former binds deeper in the cavity, forming key hydrogen-bond interactions with Q95, S56, and a water molecule. In contrast, epi-cholesterol is shifted ca. 3 Å to the mouth of the cavity and the transition to the Q95 site is prevented by an energetic barrier of 4.1 kcal·mol-1. Thus, the configuration of the hydroxyl group of cholesterol, together with the presence of a structural water molecule, is a key feature for effective absorption. Finally, whereas these findings may seemingly be challenged by single-point mutations that impair cholesterol transport but have a mild impact on the binding of cholesterol to the Q95 binding site, our results reveal that they have a drastic influence on the conformational landscape of the α8/ß7 loop in the apo species compared to the wild-type protein. Overall, the results give support to the functional role played by the α8/ß7 loop in regulating the access of ligands to NPC1L1, and hence to interpreting the impact of these mutations on diseases related to disruption of sterol absorption, paving the way to understanding certain physiological dysfunctions.

Carbon nanotube transmembrane channel formation and single-stranded DNA spontaneous internalization: a dissipative particle dynamics study.

Single-walled carbon nanotube (SWCNT) transmembrane channel formation in a pure 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) bilayer, and the spontaneous internalization of single-stranded DNA (ssDNA) into the formed pore were simulated. A combination of computational techniques, Dissipative Particle Dynamics-Monte Carlo hybrid simulations and quantum mechanical calculations at the hybrid-DFT level, was used as a new proposal to perform DPD simulations granting specific chemical identity to the model particles. The simulated transmembrane channels showed that, in the case of pristine SWCNTs and upon increasing the nanotube length, a higher tilt angle with respect to the bilayer normal is observed and more time is needed for the nanotube to stabilize. On the other hand, for SWCNTs with polar rims an almost perpendicular orientation is preferred with less than 15° of tilt with respect to the bilayer normal once the nanotubes have pierced both monolayers. These findings are supported by experimental observations where CNTs of average inner diameters of 1.51 ± 0.21 nm and lengths in the 5-15 nm range were inserted in DOPC membranes [J. Geng, et al., Nature, 2014, 514(7524), 612-615]. Moreover, the narrower the SWCNTs, the slower the spontaneous internalization of ssDNA becomes, and ssDNA ends hydrophobically trapped inside the artificial pore. A dependence on the nucleotide content is found indicating that the higher the presence of adenine and thymine in the ssDNA chains the slower the internalization becomes, in agreement with the experimental [A. M. Ababneh, et al., Biophys. J., 2003, 85(2), 1111-1127] and predicted solvation tendency in water for nucleic acid bases.