Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease

Abstract

NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two beta-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the beta-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.

FIGURE 1. Chromatographic analysis of ligand binding to NPC2

Bovine NPC2 was incubated in the absence (green) or presence of cholesterol (blue), cholesterol-3-O-sulfate (red), or cholesterol followed by cholesterol-3-O-sulfate (black) and subjected to cation exchange chromatography as described in Materials and Methods. Decreased retention time is correlated with ligand binding (12).

FIGURE 2. Electron density at the ligand-binding site

A difference electron density map (F_o − F_c) calculated at 1.8 Å resolution with phases from the final model with ligand omitted and contoured at 2.5σ reveals electron density consistent with cholesterol-3-O-sulfate within the interior of MolB (A) but not in MolA (B). The Cα traces for NPC2 MolB (blue) and MolA (green) and a stick representation of cholesterol sulfate in MolB (carbon in gold, oxygen in red, sulfur in yellow) from the final model are shown superimposed with the density maps.

FIGURE 3. Backbone displacements in apo- and sterol-bound NPC2

(A) NPC2 is shown in ribbon representation (blue) with cholesterol sulfate (carbon in gold, oxygen in red, sulfur in yellow) bound in the protein interior between strands βD and βE. The three disulfide bonds in NPC2 (yellow) and the N-acetyl glucosamine remnant of glycosylation at N39 (carbon in black, oxygen in red, nitrogen in blue) are shown in stick and ball-and-stick representation, respectively. (B) Cα traces of superimposed models of sterol-bound NPC2 (MolB, blue) and two apoNPC2 structures (MolA, green and PDB 1NEP, gold) are shown with regions of the largest displacements (rmsd >0.4 Å) highlighted in red in the model of sterol-bound NPC2.

FIGURE 4. Sterol binding cavities of apo- and sterol-bound NPC2

The ligand-binding sites in (A) apoNPC2 (PDB 1NEP), (B) apoNPC2 (MolA) and (C) cholesterol-3-O-sulfate-bound NPC2 (MolB) are shown in identical orientations in surface representation with a cross-sectioned slab removed to allow viewing of the internal ligand-binding tunnel.

FIGURE 5. Ligand-binding site residues

A Cα trace of NPC2 (blue) bound to cholesterol-3-O-sulfate (depicted as transparent spheres with carbon in white, oxygen in red, sulfur in yellow) is shown with side chains of residues that form the sterol-binding tunnel depicted in stick representation. (A) Residues that have similar positions in apo- and sterol-bound NPC2 are shown in red. Residues that occupy different positions in the closed and open states are shown in green. (B) Residues that form the binding site (blue) are colored to indicate features that reflect their malleability. Residues that have been identified in mutagenesis studies (6) to be critical to function (F66, V96, Y100) are colored red and those tolerant to non-conservative substitutions (V64, W122) are colored yellow. A residue identified in a patient with late onset disease (V20) (11) is colored orange. Residues that are not strictly conserved among mammalian NPC2 proteins (V38, V59, V64, I103, V105, I127, V129) are colored green.

FIGURE 6. Entrance to the sterol-binding tunnel

Apo- (A) and sterol-bound NPC2 (B) proteins are shown as transparent surfaces (carbon in white, oxygen in red, nitrogen in blue) with side chains of residues that form the entrance to the ligand-binding tunnel depicted in stick representation on a Cα frame. Cholesterol sulfate is shown in stick representation (carbon in gold, oxygen in red, sulfur in yellow).