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/β7 loop in the apo species compared to the wild-type protein. Overall, the results give support to the functional role played by the α8/β7 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.


TABLE OF CONTENTS
Table S1.Available structural information of the NPC1L1 protein on PDB database.S27 Table S2.Available structural information of the NPC1 protein on PDB database.S28 Table S3.Average distance (A) and preservation (%) of the interactions between wild-type NPC1L1-NTD and the studied sterol molecules.
Table S4.Description of the main conformational states of the apo and cholesterol-bound systems found for the wildtype NPC1L1-NTD and its mutated variants.

Figure S1 .
Figure S1.Structural comparison of the NTD domains of NPC1L1 and NPC1.(A) The global fold of both NTD domains of NPC1L1 and NPC1 are shown in grey and cyan cartoon respectively.(B) Comparison of the residues involved in the cholesterol binding site for both proteins.The crystallographic position of cholesterol in NPC1 is shown in orange sticks.(C) Sequence alignment between the NTDs of NPC1L1 and NPC1.Residues in the binding site of cholesterol are highlighted in cyan.

Figure S2 .
Figure S2.Superposition of the X-ray structures of the NTD domains of NPC1L1 and NPC1.(A) The global fold of both NTD domains of NPC1L1 and NPC1 are shown in grey and cyan cartoon respectively.(B) Comparison of the residues involved in the entrance of the binding site for both proteins.The crystallographic position of cholesterol in NPC1 is shown in orange sticks.

Figure S5 .
Figure S5.Selected interactions of L213 in the apo NPC1L1-NTD over simulation time.Blue and orange lines correspond to the distances of the CG atom of L213 to the CG atom of I105 and the center of the aromatic ring of F120, respectively.Green lines account for the H-bond interaction between the O atom of the backbone of L213 to the sidechain of S102.

Figure S6 .
Figure S6.Number of water molecules in the binding site of the apo state of NPC1L1-NTD over simulation time.

Figure S7 .
Figure S7.Overlap between the water network of NPC1L1-NTD with the X-ray structures of the NPC1-NTD.Close up views of the binding site in the (A) apo and (B) cholesterol-bound states.The occupancy of water molecules in the binding site over 50% of the simulation time is shown in the transparent red isosurface.The crystallographic molecules of water, cholesterol and glycerol found in the NPC1-NTD are shown as gray sticks and spheres.The NPC1L1-NTD is shown as white cartoon and sticks.The 8/7 loop is shown as blue cartoon.The residues of the binding site of NPC1L1-NTD are shown as white and blue sticks.

Figure S8 .
Figure S8.Selected interactions of L213 in the cholesterol-bound NPC1L1-NTD over simulation time.Blue and orange lines correspond to the distances of the CG atom of L213 to the CG atom of I105 and the center of the aromatic ring of F120 respectively.Green lines account for the Hbond interaction between the O atom of the backbone of L213 to the sidechain of S102.

Figure S9 .Figure S10 .
Figure S9.Number of water molecules in the binding site of the cholesterol-bound species of NPC1L1-NTD over simulation time.

Figure S11 .
Figure S11.Number of water molecules in the binding site of the epi-cholesterol-bound species of NPC1L1-NTD over simulation time.

Figure S12 .
Figure S12.Representation of the two collective variables along the well-tempered metadynamics over the Collective Variable space.CV1 accounts for the difference of distances between the O atom of epi-cholesterol and the sidechains of residues Q95 and T128.CV2 accounts for the distance between the COM of the 87 loop and the 3 helix.

Figure S13 .
Figure S13.RMSD profiles of the 25-hydroxy-cholesterol-bound NPC1L1-NTD and H-bond interactions of the sterol over simulation time.(Left) Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.(Right) Purple and orange lines correspond to the H-bonds between the hydroxyl moiety of the ligand and the sidechain of Q95 and S35.Blue lines show H-bond interaction of the 25-hydroxyl moiety.

Figure S14 .
Figure S14.RMSD profiles of the lanosterol-bound NPC1L1-NTD and H-bond interactions of the sterol over simulation time.(Left) Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.(Right) Purple and orange lines correspond to the Hbonds between the hydroxyl moiety of the ligand and the sidechain of Q95 and S35.

Figure S15 .
Figure S15.Number of water molecules in the binding site of (left) 25-hydroxy-cholesterol-and

Figure S16 .
Figure S16.RMSD profiles of apo P215A NPC1L1-NTD over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S17 .
Figure S17.RMSD profiles of apo I105A NPC1L1-NTD over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S18 .
Figure S18.RMSD profiles of apo F205A NPC1L1-NTD over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S19 .
Figure S19.RMSD profiles of apo T128A NPC1L1-NTD over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S20 .
Figure S20.RMSD profiles of cholesterol-bound P215A NPC1L1-NTD and H-bond interactions of cholesterol over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S21 .
Figure S21.RMSD profiles of cholesterol-bound I105A NPC1L1-NTD and H-bond interactions of cholesterol over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S22 .
Figure S22.RMSD profiles of cholesterol-bound F205A NPC1L1-NTD and H-bond interactions of cholesterol over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S23 .
Figure S23.RMSD profiles of cholesterol-bound T128A NPC1L1-NTD and H-bond interactions of cholesterol over simulation time.Grey and red lines correspond to the RMSD of the backbone of the protein and heavy atoms of the ligand.

Figure S24 .
Figure S24.Superposition of the major conformations of the (A,C) apo and (B,D) cholesterolbound states of the T128A mutant.(A) The major A2 and minor A1 (C) conformational states of the apo T128A mutant.(B,D) The two binding modes of cholesterol in the T128A mutants.The conformations sampled by the 8/7 loop are shown as blue cartoon.The protein surface is shown as grey surface.The isosurface accounts for 50% of water occupancy in the binding site during the last 100 ns of each replicate (accumulated 400 ns of simulation time).The water occupancy for the wildtype protein and T128A mutant are shown as grey and red isocontours, respectively.

Table S1 .
Available structural information of the NPC1L1 protein on PDB database.

Table S2 .
Available structural information of the NPC1 protein on PDB database.

Table S3 .
Average distance (A) and preservation (% of simulation time) of the interactions between wild-type NPC1L1-NTD and the studied sterol molecules.

Table S4 .
Description of the main conformational states of the apo and cholesterol-bound systems found for the wildtype NPC1L1-NTD and its mutated variants.