Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins
- Department of Biochemistry, Stanford University School of Medicine, United States
- Department of Chemistry, Technische Universität Berlin, Germany
- Chan Zuckerberg BioHub, United States
- Department of Chemical and Systems Biology, Stanford University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Locked N-terminal domain NPC1 rescues cholesterol export from lysosomes.
(A) Domain structure of NPC1 protein. The N-terminal domain (residues 23–259 including the polyproline linker), middle lumenal domain (MLD, 372–620), and C-terminal domain (CTD, 854–1098) are …
Figure 1—figure supplement 1
Characterization of selected mutant protein glycosylation status or localization.
(A) Two independent, representative experiments to analyze the glycosylation status of GFP-NPC1 wild type and GFP-NPC1 P251C/L929C, as determined by immunoblot of indicated samples with anti-GFP …
Figure 2 with 1 supplement
Engineered cysteines C251 and C929 form a disulfide bond in NPC1.
(A) Extracted ion chromatograms from LC-MS analysis of synthetic peptides corresponding to engineered cysteines in NPC1. In both samples, purple traces represent m/z = 526.2569 (corresponding to …
Figure 2—figure supplement 1
HCD mass spectra of disulfide ions (m/z = 626.60) from oxidized disulfide sample in Figure 2A.
Red peaks correspond to the y ions produced from the numbered fragmentation sites in the disulfide; cyan peaks correspond to the corresponding a and b ions. Peaks labeled ‘++’ are doubly rather than …
Figure 3
NPC1 Δloop mutant cannot rescue cholesterol export from lysosomes.
(A) Cholesterol-cross-linked peptides (Hulce et al., 2013) are highlighted in red for two orientations of the crystal structure of N-terminal domain- and first transmembrane domain-deleted NPC1 …
Figure 4 with 1 supplement
NPC1 Disulfide bond-locked MLD and CTD fails to rescue cholesterol export from lysosomes.
(A) Partial NPC1 structure; inset, close-up view of the MLD/CTD interface. The amino acid residues mutated to cysteines for disulfide bond formation are shown and highlighted in red. (B) Confocal …
Figure 4—figure supplement 1
Extracted ion chromatograms from LC-MS analysis of proteolyzed A521C/K1013C NPC1.
Protein was carbamidomethylated in the presence or absence of reducing agent prior to deglycosylation and proteolysis. In both samples, blue traces represent m/z = 596.2818 (corresponding to the …
Figure 5
Molecular dynamics simulations of NPC1 wild type and mutant proteins.
RMSD (Å) of protein backbone atoms for each simulated model is plotted as a function of time for the indicated mutants in relation to their wild type counterparts.
Figure 6
Inter-domain mobility is required for cholesterol transport by NPC1L1 protein.
(A) Structure model of NPC1L1 built by Swiss-Model using NPC1 (PDBID: 5u74) as template for UNIPROT Q9UHC9-1. Extracellular domains are labeled and colored as in Figure 1; NTD, residues 33–275; MLD, …
Figure 7 with 1 supplement
NPC1L1 MLD binds bile salt micelles.
Binding of purified NPC1L1 N-terminal domain or MLD to 3H-cholesterol, delivered either in sub-CMC NP-40 (A) or in mixed bile salt micelles (B). Error bars represent SEM for triplicate …
Figure 7—figure supplement 1
Purified NPC1L1 N-terminal domain and NPC1L1 MLD used in Figure 6.
Coomassie stained SDS PAGE gels are shown.
Figure 8
NPC1L1 N-terminal domain is essential for ezetimibe binding at the cell surface.
(A) 3H-ezetimibe binding to HEK293T cells transfected with either wild type or N-terminal domain deleted ΔN-NPC1L1. Forty-eight hours post transfection, cells were incubated with 50 nM 3H-ezetimibe …
Figure 9
Inter-domain interfaces of NPC1L1 are critical for ezetimibe binding.
(A) 3H-ezetimibe binding of HEK293T cells transfected with either wild type NPC1L1 or mutant NPC1L1 proteins designed to disrupt the interfacial interactions between domains of NPC1L1. The colored …
Tables
Table 1
Distance correlation coefficients for mutants analyzed.
Strongest inter-domain correlations are indicated in bold.
| A521C+K1013C | ||||
|---|---|---|---|---|
| NTD | MLD | CTD | TMD | |
| NTD | -- | -- | -- | -- |
| MLD | -- | 1.000 | 0.850 | 0.810 |
| CTD | -- | 0.850 | 1.000 | 0.594 |
| TMD | -- | 0.810 | 0.594 | 1.000 |
| P251C+L929C | ||||
| NTD | MLD | CTD | TMD | |
| NTD | 1.000 | 0.969 | 0.945 | 0.931 |
| MLD | 0.969 | 1.000 | 0.919 | 0.942 |
| CTD | 0.945 | 0.919 | 1.000 | 0.952 |
| TMD | 0.931 | 0.942 | 0.952 | 1.000 |
| Δ807-811 | ||||
| NTD | MLD | CTD | TMD | |
| NTD | 1.000 | 0.496 | 0.522 | 0.391 |
| MLD | 0.496 | 1.000 | 0.836 | 0.398 |
| CTD | 0.522 | 0.836 | 1.000 | 0.395 |
| TMD | 0.391 | 0.398 | 0.395 | 1.000 |
| WT | ||||
| NTD | MLD | CTD | TMD | |
| NTD | 1.000 | 0.569 | 0434 | 0.529 |
| MLD | 0.569 | 1.000 | 0.751 | 0.811 |
| CTD | 0.434 | 0.751 | 1.000 | 0.617 |
| TMD | 0.529 | 0.811 | 0.617 | 1.000 |
Table 2
Rationale for NPC1L1 domain interface mutants generated.
| Residue | Residues in other domains within 5 Å | Type of interaction | Mutation chosen |
|---|---|---|---|
| A179 | L523, Y524, N527 | Hydrophobic/hydrophilic | K (possibly trigger steric clash and disrupt the interface) |
| L427 | A180 | van der waals/hydrophobic | N (possibly disrupt the hydrophobic interface) |
| N527 | R112, Q257 | Hydrophilic | D (could stabilize the interface, restricting domain flexibility); K (potentially destabilize the interface because of positive charge repulsion) |
| K533 | K1026 | Electrostatic | E (could stabilize and restrict domain flexibility) |
| E939 | P549, F551 | van der waals | K (longer sidechain might result in steric clash, disrupt the interface) |
| R1002 | D250 | Electrostatic | E (possibly disrupt the interface) |
| K1026 | K533 | Electrostatic | R (possibly stabilize the interface and restrict domain flexibility) |
| L1029 | L530, M543 | van derwaals/hydrophobic | N (possibly disrupt the interface) |
| L987 | I72 | van derwaals/hydrophobic | N (possibly disrupt the interface) |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene Mus musculus | NPC1 | PMID:27551080 | ||
| Gene (Homo sapiens) | NPC1L1 | PMID:27075173 | ||
| Gene (Homo sapiens) | LAMP1 | PMID:27664420 | ||
| Cell line (Homo sapiens) | HeLa NPC1 KO | PMID:26578804 | ||
| Cell line (Homo sapiens) | HEK293T | ATCC | ||
| Cell line (Homo sapiens) | 293F | ATCC | ||
| Cell line (Spodoptera frugiperda) | SF9 | ATCC | ||
| Transfected construct Mus musculus | NPC1-eGFP | PMID:27551080 | ||
| Transfected construct (Homo sapiens) | NPC1L1-eGFP | PMID:27075173 | ||
| Transfected construct (Homo sapiens) | LAMP1-eGFP | PMID:27664420 | ||
| Transfected construct (Homo sapiens) | pFastBac-NPC1L1-wt-N-terminal domain | PMID:27075173 | ||
| Transfected construct (Homo sapiens) | pCMV-FLAG-His6-NPC1L1-MLD | PMID:27075173 | ||
| Antibody | Chicken polyclonal anti-GFP | Life technologies | 1:1000 | |
| Antibody | IRDye 680RD polyclonal Donkey anti-chicken | LI-COR | 926–68075 | 1:10000 |
| Antibody | IRDye 800CW streptavidin | LI-COR | 926–32230 | 1:10000 |
| Antibody | Mouse monoclonal anti-GFP | NeuroMab | N86-38 | 1:1000 |
| Antibody | rabbit polyclonal anti-LAMP1 | Novus | NB120-19294 | 1:1000 |
| Antibody | Alexa Fluor 488 polyclonal Goat anti-mouse | Life Technologies | A-11001 | 1:2000 |
| Antibody | Alexa Fluor 568 polyclonal Goat anti-rabbit | Life Technologies | A-11011 | 1:2000 |
| Recombinant DNA reagent | 293 fectin | Invitrogen | ||
| Recombinant DNA reagent | polyethyleneimine | SIGMA-Aldrich | ||
| Recombinant DNA reagent | Lipofectamine 3000 | Life Technologies | Cat #A10262 | |
| Chemical compound, drug | Ezetimibe | Santa Cruz Biotechnology | sc-205690 | |
| Software, algorithm | Flowjo | https://www.flowjo.com/ | ||
| Software, algorithm | FIJI | PMID:22743772, doi:10.1038/nmeth.2019 |
