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. 2017 Aug 22;114(34):9116-9121.
doi: 10.1073/pnas.1711716114. Epub 2017 Aug 7.

3.3 Å structure of Niemann-Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

Xiaochun Li  1   2 , Feiran Lu  3 , Michael N Trinh  3 , Philip Schmiege  4   2 , Joachim Seemann  5 , Jiawei Wang  6 , Günter Blobel  1   2
Affiliations

3.3 Å structure of Niemann-Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

Xiaochun Li et al. Proc Natl Acad Sci U S A. .

Abstract

Niemann-Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann-Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314-1,278), which-in contrast to previous lower resolution structures-features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909-C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD-NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

Keywords: Niemann–Pick type C disease; cholesterol transport; crystal structure; cysteine-rich domain; sterol-sensing domain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structure of the NPC1 protein and structural details of its CTD. (A) Schematic representation of NPC1 domains with the part after the proteinase K site (indicated by “Δ”) crystallized here in color. (B) The overall structure of NPC1* with TM2–7 colored blue, TM8-13 in green, MLD in pink, and CTD in light purple. (C) Structure of the NPC1-CTD with the secondary elements labeled and the cysteine side-chain atoms involved in disulfide bonds shown as balls with different colors. (D) Primary structure of the CTD, with the major structural elements indicated above the sequence and disulfide bonds indicated by colored lines (colored as in C).
Fig. S1.
Fig. S1.
The 2Fo-2Fc electron density of NPC1*. (A) An overall view of the 2Fo-2Fc electron density map, contoured at 1.0 σ, in one unit cell. (B) A representative view of the 2Fo-Fc electron density map at various regions, contoured at 1.0 σ.
Fig. S2.
Fig. S2.
Structural comparison of the MLD and the CTD. (A) Structures of the MLD (pink) and the CTD (light purple) are superimposed with the major elements of two domains indicated; disulfide bonds of the CTD are shown as balls with different colors as in Fig. 1C; disulfide bonds of the MLD are shown as balls in teal. (B) Close-up view of α2 of the both domains.
Fig. 2.
Fig. 2.
The interface of NPC1-CTD and NPC1-NTD. (A) Docking of NPC1* to cryo-EM NPC1 structure (PDB ID code 3JD8); the NTD and TM1 from EM structure are presented with the 3.3 Å NPC1* crystal structure; cholesterol molecule is indicated as stick. (B) Close-up view of the interaction between the NPC1-CTD and NPC1-NTD; residues involved in this interaction are labeled and hydrogen bonds are indicated by dashed lines. The disulfide bonds are indicated by sticks and colored as Fig. 1 C and D; the proline-rich region is indicated; the CTD is colored in gray except for the light purple region that interacts with the NTD.
Fig. S3.
Fig. S3.
Structural comparison of NPC1* crystal structure and cryo-EM structure of full-length NPC1.
Fig. S4.
Fig. S4.
Comparison of NPC1* crystal structure and NPC1 cryo-EM structure (PDB ID code 3JD8). The structure of NPC1* is colored in purple, and the cryo-EM structure is colored in cyan; the map of cryo-EM (EMD-6640) is shown as gray surface at the 5.0 σ level.
Fig. 3.
Fig. 3.
Specific residues of the NPC1-CTD are required for cholesterol esterification. (A) Confocal immunofluorescence microscopy analysis of the localization of NPC1ΔΩ-A, NPC1Δψ, and LAMP2 proteins in NPC1−/− 10–3 cells 24 h after transfection as described in Experimental Procedures. (Scale bar, 20 µm.) (B) Cholesterol esterification: After labeling as described in Experimental Procedures, cells were harvested for measurement of their content of cholesteryl [14C]oleate and [14C]triglycerides. Each value is the mean of duplicate incubations with individual values shown. The cellular content of [14C]triglycerides in all transfected cell lines did not differ significantly in cells treated with LPDS (8–11 nmol·h–1·mg–1) or FCS (13–15 nmol·h–1·mg–1). The immunoblot analysis of whole cell extracts (6 µg) from the indicated transfection shown below was done using 0.5 µg/mL NPC1 antibody and 2 µg/mL NPC2 antibody as a loading control.
Fig. S5.
Fig. S5.
Structural prediction of NPC1ΔΩ-A. (A) The localization of Ω loop in the NPC1 (red), with the CTD colored in light purple. (B) Close-up view of CTD-α5 structure in the NPC1-WT (red). (C) Close-up view of CTD-α5 model in the NPC1ΔΩ-A(red).
Fig. 4.
Fig. 4.
The specific location of I1061 and P691 in NPC1. (A) The location of I1061. Close-up view of 2Fo-Fc density map of I1061 (black mesh) contoured at σ level 1.0. (B) The location of P691. Close-up view of 2Fo-Fc density map of P691 (black mesh) contoured at σ level 1.0. The putative ligand-binding pocket of the SSD is indicated by a black oval.
Fig. 5.
Fig. 5.
The distribution of NPC-causing mutations. The overall structure rotated by 90° with balls at each mutated residue. Color code for mutations: cyan, NTD and TM1; blue, TMs 2–7; pink, MLD; green, TMs 8–13; and light purple, CTD.
Fig. 6.
Fig. 6.
Models of NPC2–NPC1-mediated cholesterol transfer. The Ω loop (red) might keep the NTD in the proper orientation for receiving the cholesterol from NPC2 when NPC2 binds to the MLD. Cholesterol (green balls) is transferred from NPC2 (yellow) to NPC1-NTD (cyan) when NPC2 binds to NPC1-MLD (magenta). After this transfer, the NTD could reorient across the glycocalyx to dock in the gap between the SSD (blue) and the MLD for delivering the cholesterol to the SSD. The mechanism of this step is still unclear. Two possibilities exist: (1) The first is intramolecular transfer. Because the NTD is far away from the SSD, a conformational change is required for intramolecular transfer. The long linker between TM1 (cyan) and TM2 (on the edge of the SSD) might induce movement of TM1 to allow the proximity between NTD to SSD in one NPC1 molecule to trigger this transfer. (2) The other is intermolecular transfer. The NTD of one NPC1 molecule could insert into another NPC1-SSD without movement of TM1 and without any large conformational change. Black arrows indicate the transfer orientation of the cholesterol molecule. The P691 in the SSD cavity is indicated in red.
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