The structure of the NPC1L1 N-terminal domain in a closed conformation

Abstract

Background: NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels.

Principal findings: Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent.

Conclusion: The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.

Figure 1. Sterol Binding and Specificity of NPC1L1(NTD).

(A) Cholesterol binding. Each reaction, in a final volume of 100 µl, contained 0.5 pmol of purified HIS6-NPC1L1(NTD) and the indicated concentration of ³H-cholesterol in the absence (•) or presence (○) of 100 uM unlabeled cholesterol. Bound ³H-cholesterol was measured as described in Materials and Methods . Each data point represents the average of triplicate assays. (B) Competitive binding of ³H-cholesterol in the presence of unlabeled sterols. Each reaction, in a total volume of 100 µl, contained 0.5 pmol HIS6-NPC1L1(NTD), 10 nM ³H-cholesterol and 1 µM of the indicated unlabeled sterol. Each data point represents the average of triplicate assays and represents the amount of ³H-cholesterol bound relative to that in the control tube, which did not contain unlabeled sterol. (C) Competitive binding of ³H-cholesterol in the presence of unlabeled sterols. Each reaction, in a total volume of 100 µl, contained 0.5 pmol HIS6-NPC1L1(NTD), 10 nM ³H-cholesterol and varying concentrations of the indicated unlabeled sterol. Each data point represents the average of triplicate assays and represents the amount of ³H-cholesterol bound relative to that in the control tube, which did not contain unlabeled sterol.

Figure 2. The Structure of NPC1L1(NTD).

(A) NPC1L1(NTD) is represented as a ribbon diagram and the disulfide bonds are shown in yellow. Domain A is colored orange (α-helices), cyan (β-sheets), and gray (loops). α3 (red), α7 (blue), and the α8/β7 loop (green) surround the entrance of the cholesterol binding pocket. Domain B is colored magenta. (B) Superposition of NPC1L1(NTD) (yellow) and NPC1(NTD) (gray). Bound cholesterol in NPC1 is shown as a stick model. Regions around the entrance of the cholesterol binding pocket in NPC1L1 are colored red (α3), blue (α7), and green (α8/β7 loop). (C) Sequence alignment of NPC1L1(NTD) and NPC1(NTD). N-linked glycosylation sites are shaded gray. Residues lining the cholesterol binding pocket are shaded yellow in NPC1. In NPC1L1, residues within the interior of the closed cholesterol binding pocket are shaded blue, residues on the exterior of the closed cholesterol binding pocket are shaded green, and residues separating the interior from the exterior are shaded red. Regions around the entrance to the cholesterol binding pocket that change conformation are boxed. The secondary structure of NPC1L1(NTD) is shown below the sequence.

Figure 3. NPC1L1(NTD) in a closed conformation.

(A) The surface of NPC1L1(NTD), colored green, reveals a cholesterol binding pocket (blue) that is closed to solvent. (B) The surface of NPC1(NTD), colored gray, shows the cholesterol binding pocket (yellow) exposed to solvent. The isooctyl side chain of cholesterol (stick model in green) is visible and solvent exposed. (C) Cutaway view of the cholesterol binding pocket of NPC1L1(NTD) in the closed conformation. (D) Cutaway view of the cholesterol binding pocket of NPC1(NTD) in an open conformation.

Figure 4. Structural differences between NPC1L1 and NPC1.

(A) In the closed form of NPC1L1(NTD), ASN211 forms a hydrogen bond with ASP208, allowing LEU213 to rotate toward α7. (B) In the cholesterol bound form of NPC1(NTD), ASN198 forms a hydrogen bond with the main chain amine of GLN200, which is rotated away from α7. (C) Superposition of NPC1L1(NTD) (yellow) and NPC1(NTD) (gray) within the cholesterol binding pocket. The smaller residues of NPC1L1, PHE35 and LEU216, expand the cholesterol binding pocket, accommodating sterols such as lanosterol.