Comparative study of the effect of disease causing and benign mutations in position Q92 on cholesterol binding by the NPC1 n‐terminal domain

Abstract

The Niemann-Pick type C1 (NPC1) protein is a large transmembrane protein located in lysosomes/endosomes. NPC1 binds cholesterol (CLR) and transports it to cellular membrane and endoplasmic reticulum. Mutations in NPC1 cause Niemann-Pick type C (NPC) disease, a rare autosomal disorder characterized by intracellular accumulations of CLR and subsequent neurodegeneration leading to premature death. Among known disease-causing mutations in NPC1, Q92R is the one that is located in the N-terminal cholesterol-binding domain [NTD]. Here we study the effect of the mutation on the ability of NPC1 (NTD) to bind and retain CLR in the binding pocket using structural analysis. We compare characteristics of the Q92R and Q92S mutant type (MT) protein, which is predicted to be benign. We provide detailed investigation of the CLR-NPC1 (NTD) binding process; and propose the mechanism, by which Q92R mutation causes NPC disease. We show that although Q92 residue neither directly participates in catalytic activity of the NPC1 (NTD), nor defines its CLR-binding specificity - it is important for the overall protein structure as well as for providing favorable electrostatic environment for CLR transfer. Our results suggest that a negative electrostatic potential of the CLR binding site (the S-opening) might promote NPC2 interaction with NPC1 (NTD) and/or proper CLR orientation and its enforced transfer. We show that in contrast to the benign Q92S mutation, Q92R significantly reduces electrostatic potential around S-opening, and thus likely affects NPC1 (NTD)-NPC2 interaction and/or CLR transfer from NPC2 to NPC1.

Signature of N-terminal domain (NTD) structural re-orientation in NPC1 for proper alignment of cholesterol transport: Molecular dynamics study with mutation

“…[35] On the other hands, the computer simulation with NTD protein and its two mutations (Q92R and Q92S) shows the importance of correct electrostatic distribution near the entrance of cholesterol pocket as well as structural stability for suitable binding cholesterol to NTD. [36] In combination with experiment, the molecular dynamics simulation of L472P mutation indicates disruption of the tunnel between MLD and CTD that is believed to play essential role for cholesterol transport as mentioned earlier. [27] Since it was pointed that the R518W mutation decrease cholesterol transfer activity not because of misfolding of NPC1 but because of the functional defection of NPC1, [12] the atomic level detailed mechanism behind this defective functionality could provide insights on the structure and functional relationship of NPC1, especially its interaction with NPC2.…”

Computational Modeling Explains the Multi Sterol Ligand Specificity of the N-Terminal Domain of Niemann–Pick C1-Like 1 Protein

“…Those residues have been implicated in control of the sterol transfer between NPC1–NTD and NPC2. ,, However, it is important to note that there are also residues in NPC1–NTD whose mutation leads to an NPC disease phenotype and which have no counterparts in NPC1L1–NTD. For example, Q92, which corresponds to A108 in NPC1L1–NTD, is located next to the pocket entrance (Figure S12), where it stabilizes the opening and overall structure of the NPC1–NTD binding pose . Mutations Q92R and Q92S in NPC1–NTD are known to give a severe clinical phenotype. , It is likely that the majority of these nonconserved residues leading to NPC disease is involved in docking to or sterol transfer from NPC2 or plays other functional roles apart from sterol binding.…”

Effect of pH on the Ability of N-Terminal Domain of Human NPC1 to Recognize, Bind, and Transfer Cholesterol

“…RMSD/RMSF Analysis. Once the production phase of MD simulations for each of the systems was finished, the snapshots were aligned in a way to minimize RMSF of all cysteine residues within the snapshot 26 (as they were shown to be important in supporting the protein structure by forming cysteine bridges 8 ). Aligned snapshots allowed generating the average protein structure.…”

“…NPC2-NPC1 interaction promotes the CLR transfer from NPC2 to NPC1 by facilitating reorientation of the helical subdomain, enlarging opening (“S-opening”) of the CLR-binding pocket in NPC1 and thus releasing CLR. ,,,, The type of interaction between NPC1 and NPC2 for CLR transport was termed a “hydrophobic hand-off” or “sliding” transfer model, where close NPC2-NPC1 interaction allows CLR transfer from NPC2 to NPC1­(NTD) without being exposed to the water phase. ,− NTD binds the 3β-hydroxyl site of CLR facing the interior of the domain and the iso-octyl side chain exposed, which is opposite to orientation of CLR when bound by NPC2. ,, NPC1­(NTD) is located in the luminal part of endosomes/lysosomes, , where pH is in a range of 4.0–6.5 − and CLR transport from NPC2 to NPC1 is pH dependentit is twice slower at pH = 7.0 compared to pH = 5.5 . Previously, we demonstrated that the Q92 residue in NTD is crucial in CLR transfer from NPC2 to NPC1 as it provides the favorable electrostatic environment between proteins . Both MLD and CTD interact with the NTD and thus modulate its activity. , …”

“…11 Previously, we demonstrated that the Q92 residue in NTD is crucial in CLR transfer from NPC2 to NPC1 as it provides the favorable electrostatic environment between proteins. 26 Both MLD and CTD interact with the NTD and thus modulate its activity. 12,22 Although many attempts have being made to crystallize the complete structure of NPC1, the large size of the protein and the fact that it has a substantial transmembrane component are challenges for X-ray crystallography.…”