Molecular dynamics study with mutation shows that N-terminal domain structural re-orientation in Niemann-Pick type C1 is required for proper alignment of cholesterol transport

Abstract

The lysosomal membrane protein Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) are main players of cholesterol control in the lysosome and it is known that the mutation on these proteins leads to the cholesterol trafficking-related neurodegenerative disease, which is called the NPC disease. The mutation R518W or R518Q on the NPC1 is one of the type of disease-related mutation that causes cholesterol transports to be cut in half, which results in the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment of the cell. Even though there has been significant progress with understanding the cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of the full-length NPC1 in 2016, many details such as the interaction of the full-length NPC1 with the NPC2, the molecular motions responsible for the cholesterol transport during and after this interaction, and the structure and the function relations of many mutations are still not well understood. In this study, we report the extensive molecular dynamics simulations in order to gain insight into the structure and the dynamics of NPC1 lumenal domain for the cholesterol transport and the disease behind the mutation (R518W). It was found that the mutation induces a structural shift of the N-terminal domain, toward the loop region in the middle lumenal domain, which is believed to play a central role in the interaction with NPC2 protein, so the interaction with the NPC2 protein might be less favorable compared to the wild NPC1. Also, the simulation indicates the possible re-orientation of the N-terminal domain with both the wild and the R518W-mutated NPC1 after receiving the cholesterol from the NPC2 that align to form an internal tunnel, which is a possible pose for further action in cholesterol trafficking. We believe the current study can provide a better understanding of the cholesterol transport by NPC1 especially the role of NTD of NPC1 in combination with NPC2 interactions.

Keywords: COVID-19; NPC1; NPC2; R518W mutant; cholesterol trafficking; molecular dynamics simulation; neurodegenerative Niemann-Pick C disease.

Figure 1

The initial structure we have prepared for simulations, including the cholesterol in the NTD. The cholesterol is shown as a gray sphere. The NTD, C‐terminal lumenal domain (CTD), and MLD domains are shown as green, magenta, and cyan, respectively. The yellow stick is the Arg518 residue which is subject to the mutation of the Trp in our simulation. The Niemann‐Pick type C2 binding loops are shown in black and the five restrained C _α atoms are shown as spheres in a wheat color

Figure 2

The time evolution of the angles among the NTD, MLD, and C‐terminal lumenal domain (CTD) for three trajectories. Panel a corresponds to the trajectory with the titled NTD. We selected a helix from each domain and calculated the angles between each helix axis. Please refer to Figure S2 for the selected helices in each domain. The black, red, and green lines correspond to the angle between NTD and MLD, angle between MLD and CTD, and angle between CTD and NTD, respectively. The angles between each domain are obtained based on a specific helix as an axis from each domain

Figure 3

The structure obtained from the current simulation with a wild type after 0.2 μs (a), 0.5 μs (b), and 1.0 μs (c) that is in overlapped with the initial structure (yellow). The color of each domain is same as the color used in Figure 1

Figure 4

Fitting of (a) the “Texas” model (lime) and (b) the “California” model (yellow) onto the structure of the WT Niemann‐Pick type C1 (NPC1) obtained from the current simulation without cholesterol (blue). The coordinate of “Texas” model is from Estiu et al. (Estiu et al., 2013). We obtained the “California” model structure following the procedure in the reference (Li, Saha, et al., 2016). Namely, we overlapped Niemann‐Pick type C2 (NPC2) binding X‐ray structure (PDB ID: 5KWY) onto cryo‐EM structure (PDB ID: 3JD8). Then, we aligned the two NPC2 binding loops on MLD in 5KWY to the same loops of 3JD8 along with the NPC2 in 5KWY. The structure in figure (a) is a result from combination of the NTD/NPC2 complex having the “Texas” interface with full‐length NPC1 from current simulation. If the “Texas” model is reasonable, this is a putative structure of full‐length NPC1 in complex with cholesterol bound NPC2 for the cholesterol transport from NPC2 to NTD. This model may serve as a template for further study such as monitoring the relative orientation/distance change right after NPC2 binding in MLD loops

Figure 5

The overlap of the structure obtained from the mutation simulation over the X‐ray structure of the Niemann‐Pick type C2 (NPC2) that is in binding with the MLD. It generates some structural crashes near the interface between NPC2 and NTD. The detailed structure of the interface is shown along with the residues. The mutated residue Trp518 was presented as a red stick for convenience. For clarity, the same structure from the rear side is shown as the magnified figure on the right hand side

Figure 6

The RMSD time profile of the wild‐type Niemann‐Pick type C1 (NPC1) simulation with cholesterol in the NTD. The RMSD of the whole system is shown as black line. The RMSD of NTD, MLD, and C‐terminal lumenal domain (CTD) are shown as red, green, and blue lines, respectively

Figure 7

The change in the angles as a function of time for a wild‐type trajectory with cholesterol in the NTD. The angles here are obtained using the same method as in Figure 2

Figure 8

The tunnel obtained from the wild‐type cholesterol containing an NTD simulation. The tunnel is identified with MoleOnline (Berka et al., 2012) and is represented as black mesh. The NTD, C‐terminal lumenal domain (CTD), and MLD domains are shown as green, magenta, and cyan, respectively

Figure 9

The same Figure as Figure 9 with an R518W mutation with cholesterol in the NTD. The structure shown is extracted at 200 ns. The NTD, C‐terminal lumenal domain (CTD), and MLD domains are shown as green, magenta, and cyan, respectively