Computational Modeling Explains the Multi Sterol Ligand Specificity of the N-Terminal Domain of Niemann–Pick C1-Like 1 Protein

Abstract

Niemann–Pick C1 like 1 (NPC1L1) is a sterol transporter expressed in the apical membrane of enterocytes and hepatocytes. NPC1L1 resembles the lysosomal NPC1 protein including an N-terminal domain (NTD), which binds a variety of sterols. The molecular mechanisms underlying this multiligand specificity of the NTD of NPC1L1 (NPC1L1–NTD) are not known. On the basis of the crystal structure of NPC1L1–NTD, we have investigated the structural details of protein–sterol interactions using molecular mechanics Poisson Boltzmann surface area calculations here. We found a good agreement between experimental and calculated binding affinities with similar ranking of various sterol ligands. We defined hydrogen bonding of sterol ligands via the 3′-β-hydroxy group inside the binding pose as instrumental in stabilizing the interaction. A leucine residue (LEU213) at the mouth of the binding pocket transiently opens to allow for the access of sterol into the binding pose. Our calculations also predict that NPC1L1–NTD binds polyene sterols, such as dehydroergosterol or cholestatrienol with high affinity, which validates their use in future experiments as close intrinsically fluorescent cholesterol analogs. A free energy decomposition and computational mutation analysis revealed that the binding of various sterols to NPC1L1–NTD depends critically on specific amino acid residues within the binding pocket. Some of these residues were previously detected as being relevant for intestinal cholesterol absorption. We show that clinically known mutations in the NPC1L1–NTD associated with lowered risk of coronary heart disease result in strongly reduced binding energies, providing a molecular explanation for the clinical phenotype.

Binding free energy of several sterols to the N‐terminal domain of Niemann‐Pick C1‐like 1 protein due to mutation: Molecular dynamics study

“…We note that these simulation time lengths are considerably longer than the simulation time that Poongavanam et al have used in their free energy estimation. [ 12 ] The final structures of each system from the simulations are shown in Figure 4.…”

“…For the charge calculation of the sterol molecules, the electronic structure calculation was performed for each sterol molecule, and the optimized structure was obtained using Gaussian 09 [21] software at the level of HF/6-31G** and the atomic partial charges were obtained at the level of B3LYP/cc-pVTZ using the optimized structures. [12] An implicit water environment IEF-PCM continuum solvation model [22,23] was utilized during this electronic structure calculation. The pressure was set to 1 bar using a Berendsen barostat with a coupling constant of 2 ps.…”

“…[ 11 ] As for the NPC1L1, Poongavanam et al estimated the change of cholesterol binding free energies in several computational simulations due to point mutations of NPC1L1‐NTD (T61M, I105M, L110M, T128A, N132S, F205A, P215A, L216A). [ 12 ] They observed that compared to wild NTD, N132S and F205A showed high binding affinity while the remainder showed low binding affinity. Zhang et al suggested a ligand that can bind both pancreatic triglyceride lipase (PTL), a therapeutic target for obesity, and NPC1L1, based on a one‐step modification of existing blockers using docking.…”

NPC1L1 Plays a Novel Role in Nonalcoholic Fatty Liver Disease

“…NPC1L1 is a transmembrane protein consisting of 1332 amino acids with 13 transmembrane domains and a wide range of glycosylation sites including an NPC domain and a sterol sensing domain (SSD). , Altman confirmed that NPC1L1 was located on the brush marginal membrane of intestinal epithelial cells and mainly located in jejunal epithelial cells using immunocombination and in situ hybridization techniques . NPC1L1 plays an important role in the sterol lipid metabolism pathway and is a key protein for the intestinal absorption of sterol lipids, especially cholesterol.…”

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

“…NPC1L1 imports selectively cholesterol as well as sterol precursors and vitamins . The molecular basis of its ligand specificity is encoded into the structural features of the NTD binding site. , The X-ray structure of the apo NTD has been solved in its closed conformation, which prevents access to the binding site via the steric blockage by the side chain of L213 of the α8/β7 loop (Figure B). Although there is no X-ray structure for the cholesterol-bound NTD, details of the binding mode have been gained from the holo species of the homologous Niemann–Pick C1 (NPC1) protein (40% identity and 57% similarity; 41% identity and 64% similarity considering only the residues involved in the cholesterol-binding site), which has been solved in complex with cholesterol and 25-hydroxycholesterol .…”