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• W4248892569 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1’s N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, ezetimibe, blocks NPC1L1; we show that residues that lie at the interface between NPC1L1's three extracellular domains comprise the drug’s binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and ezetimibe blocks transport by binding to multiple domains simultaneously. Introduction NPC1 and NPC1L1 are structurally related, multi-spanning membrane proteins that are important for cholesterol transport in humans. NPC1L1 mediates the uptake of dietary cholesterol at the surface of the intestinal epithelium (Altmann et al., 2004; Weinglass et al., 2008; Jia et al., 2011); the drug, ezetimibe (Zetia) blocks NPC1L1 and is an alternative to statins for patients with elevated plasma cholesterol (Rosenblum et al., 1998; van Heek et al., 2000; Davis et al., 2001). The related NPC1 protein functions in lysosomes to transport LDL-derived cholesterol to the cytoplasm (Pfeffer, 2019). Plasma LDL is delivered to the lysosome by endocytosis, and its cholesterol esters are cleaved by acid lipase to release free cholesterol for cellular use (Brown and Goldstein, 1986; Goldstein et al., 1975). NPC2 protein binds this released cholesterol via its iso-octyl group (Xu et al., 2007) and transfers it to the N-terminal domain of NPC1 (Kwon et al., 2009). Mutations in either NPC2 or NPC1 protein can give rise to a severe neurodegenerative disorder called Niemann Pick Type C disease, which leads to massive accumulation of cholesterol in lysosomes of all tissues, and premature death (Pentchev, 2004). The availability of the structures of NPC2 (with and without cholesterol; Friedland et al., 2003 and Xu et al., 2007), NPC1 N-terminal domain (Kwon et al., 2009), lumenal domains 2 and 3 (hereafter referred to as middle lumenal domain/MLD and C-terminal domain/CTD; Li et al., 2016a; Li et al., 2017b) and the full length NPC1 by cryoelectron microscopy (Gong et al., 2016; Long et al., 2020) has catapulted our understanding of these proteins to a new level. Moreover, the co-crystal structure of NPC2 bound to NPC1’s second lumenal domain (Li et al., 2016b; see also Winkler et al., 2019) provides a valuable starting point for thinking about how cholesterol is likely to be transferred onto NPC1 protein. Despite these important breakthroughs, we still have much to learn about how NPC1 transfers cholesterol across the lysosome membrane after receiving it from NPC2. It has been proposed that the NPC1 N-terminal domain uses the flexibility of a poly-proline linker to transfer cholesterol to a cavity detected on the other side of the protein, at the so-called sterol-sensing domain (Kwon et al., 2009; Li et al., 2016a; Li et al., 2017b). This cavity lies at the boundary between the inner leaflet of the lysosome membrane and the lumen, an advantageous position as the cavity would be available to both receive cholesterol from NPC1’s N-terminal domain and to transfer it to the adjacent membrane. Consistent with this model, Trinh et al. (2018) recently found that NPC1’s N-terminal domain appears to be able to transfer cholesterol (albeit inefficiently) to an adjacent NPC1 molecule for membrane transfer. NPC1 domains that receive cholesterol from NPC2 are located ~80 Å from the membrane bilayer (Gong et al., 2016). This height corresponds well with the dimensions of the glycocalyx that is thought to line the limiting membrane of the lysosome (Wilke et al., 2012). Given the existence of the glycocalyx, it is hard to imagine how NPC1’s N-terminal domain could gain access to the lipid bilayer to deliver cholesterol to the membrane. In considering the established mechanisms of transporters for amino acids, sugars, and hydrophobic small molecules, we noted that these extremely diverse proteins undergo significant conformational changes to enable an open extracellular pocket to bind ligand and then close, thereby opening a release site on the opposite side of the protein (and membrane). This type of ‘rocker arm’ model would require movement of protein domains in relation to one another, for transport functionality. We therefore set out to test whether NPC1 functions by conformational transformations rather than via a sterol hand-off pathway restricted to the N-terminal domain. We present here data consistent with a model in which cholesterol passes directly through NPC1 and NPC1L1 proteins and can be blocked by plugging the channel at the top with a small molecule inhibitor such as ezetimibe for NPC1L1 (this study) or itraconazole for NPC1 (Long et al., 2020). Results Figure 1A shows the overall domain structure of NPC1 protein, which comprises a cholesterol binding N-terminal domain (red), a second lumenal domain that binds to NPC2 (‘MLD’, blue; Deffieu and Pfeffer, 2011; Li et al., 2016b) and a third lumenal domain (‘CTD’, yellow). The (red) N-terminal domain is attached to the rest of the protein by a potentially flexible polyproline linker sequence (Figure 1B at left). Note that the putative sterol-binding site in the sterol-sensing domain (SSD, orange) is located within the membrane bilayer on the side of the protein opposite to the linker domain, adjacent to P691, a residue that is important for NPC1 function (Watari et al., 1999; Ko et al., 2001; Ohgami et al., 2004). To test whether locking the flexible linker in place would inhibit NPC1 function, we relied on the highest resolution (3.3 Å) NPC1 structure (Li et al., 2017b) to introduce cysteines that could pair between the top of the polyproline linker and the NPC1 CTD. To design these experiments, we aligned the cryo-EM structure of full-length NPC1 (4.4 Å, PDBID: 3jd8; Gong et al., 2016) with the high resolution (3.3 Å) crystal structure of N-terminal domain-deleted NPC1 (PDBID: 5u74 (Li et al., 2017b; Figure 1B). As shown in Figure 1C, introduction of two cysteine residues at positions P251 (in the linker) and L929 (Figure 1B inset) did not interfere with the proper folding of NPC1 and its proper delivery to lysosomes as monitored by immunofluorescence microscopy and co-localization with LAMP1. Figure 1 with 1 supplement see all Download asset Open asset 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 colored red, blue, and yellow, respectively. (B) NPC1 residues mutated to Cys for disulfide bond formation between the polyproline linker and CTD (see inset). The location of the sterol-sensing domain is shown in orange; P691 faces the back. (C) Confocal immunofluorescence microscopy of mouse NPC1 P251C/L929C and LAMP1 proteins expressed in HeLa cells (bar, 20 μm). (D) Confocal immunofluorescence microscopy of cholesterol accumulation rescue. NPC1−/− HeLa cells were transfected with GFP-mouse NPC1-wild type or P251C/L929C plasmids for 48 h. Thirty-two hours post transfection, cells were incubated with 1 µM U18666a for 16 h; cells were briefly incubated with 10 mM methylamine hydrochloride and chased for cholesterol export for 1 h in 5% LPDS medium, followed by immediate fixation. Intrinsic GFP fluorescence and AF647-PFO* labeling are shown (bar, 20 μm). Images represent maximum intensity projections. (E) Flow cytometry of the experiment shown in (D). GFP-positive cells of similar intensity were analyzed: NPC1-/-, 497 cells; NPC1 wild type, 478 cells; P251/L929C, 1486 cells. Cell numbers were normalized for comparison. (F) Flow cytometry of a rescue experiment using the indicated constructs, carried out as in (E). GFP-positive cells were analyzed: NPC1-/-, 2968 cells; NPC1 wild type, 2358 cells; P251/L929C/P249K/P259K, 3906 cells. Flow cytometry analyzed only GFP-positive cells of similar intensity. Perfringolysin O* (PFO*) is a protein that binds cholesterol and can be used to quantitate cholesterol levels in cells lacking NPC1 protein (Das et al., 2013; Li et al., 2015). The protein is tagged with a fluorescent dye and can be detected by quantitative flow cytometry or light microscopy to monitor lysosomal cholesterol. As shown in Figure 1D, NPC1-/- cells lacking transfected NPC1 showed strong PFO* staining; transient expression of wild type NPC1 protein (green) rescued the phenotype and could be easily quantified as a decrease in PFO* signal. To compare the activities of wild type and mutant proteins more sensitively, we attempted to compare the initial rates of cholesterol clearance by functional or non-functional NPC1 upon expression in NPC1 knockout cells. For this purpose, we synchronized the cholesterol release process in cells expressing rescue constructs by addition of the NPC1-specific inhibitor, U18666A (Lu et al., 2015) and then monitored the cholesterol export upon removal of the inhibitor. Protein constructs were expressed for ~30 h, followed by overnight treatment of cells with U18666A; we then washed out U18666A and monitored the subsequent relative function of rescue constructs after 1 h, as this short time scale allows for better functional discrimination than is possible when comparing cholesterol accumulation by constructs expressed for 18–24 h. U18666A drug efflux was accelerated by treating cells for 5 min in 10 mM methylamine hydrochloride to raise the pH of endo-lysosomes. The pH was then quickly restored by methylamine washout, and NPC1 function was monitored after 1 subsequent hour in lipoprotein-deficient medium. Control experiments showed that wild type NPC1 required at least 30 min to begin to decrease lysosomal cholesterol by this method. Using this assay, we found that NPC1 P251C/L929C was as functional as wild type NPC1 in terms of its ability to clear cholesterol from lysosomes, monitored by quantitative flow cytometry or immunofluorescence light microscopy (Figure 1D,E). Note that the flow cytometry data have been gated such that only cells with comparable levels of expression of wild type or mutant GFP-tagged proteins are compared. Critical to the interpretation of these data was a demonstration that a disulfide bridge had formed between residues C251 and C929. We noted that NPC1 P251C/L929C was more efficiently glycosylated in the secretory pathway than either the wild type protein or a single cysteine mutant, consistent with formation of this disulfide bond within the secretory pathway of expressing cells (Figure 1—figure supplement 1A). Mature and fully glycosylated NPC1 protein migrates at ~200K upon SDS-PAGE; with endo H treatment to remove high mannose oligosaccharides, a slight mobility decrease is observed (Figure 1—figure supplement 1). Less well-folded NPC1 proteins are fully endo H sensitive and yield a band at ~120K upon endo H or protein N-glycanase treatment that may be O-glycosylated (Schultz et al., 2018). In our cells, under-glycosylated NPC1 wild type protein was detected, but much less of this form was seen for the P251C/L929C protein. That we detect differences in the steady state glycoforms of NPC1 proteins indicates that they are structurally distinct in cells, consistent with the presence of the additional disulfide bond. To demonstrate unequivocally the presence of a disulfide bond between NPC1 residues C251 and C929, we used mass spectrometry to detect the disulfide bond in proteolytic digests of the GFP-tagged protein, isolated after expression in cultured cells. Unfortunately, it was difficult to identify the native peptides because of their lengths and polyproline content. To circumvent this challenge, we introduced two lysine residues adjacent to the polyproline stretch in NPC1 P251C/L929C to enhance trypsin cleavage and facilitate disulfide bond detection. Figure 1F shows the cholesterol rescue activity of NPC1 P251C/L929C/P249K/P259K. Importantly, this NPC1 mutant was correctly localized to lysosomes (Figure 1—figure supplement 1B) and showed full wild type rescue capacity by quantitative perfringolysin O* labeling (Figure 1F). Proteolysis of NPC1 P251C/L929C/P249K/P259K would be predicted to yield disulfide-bonded peptides, with C251 in CQPPPPPMK linked to C929 in NAAECDTY. Figure 2A shows the LC-MS elution profiles of both reduced (upper panel) and chemically oxidized (lower panel) forms of synthetic peptides corresponding to these NPC1 peptides (see also Figure 2—figure supplement 1). Guided by these peptide standards, we detected the corresponding disulfide species in NPC1 P251C/L929C/P249K/P259K samples expressed in cultured cells followed by protein purification and protease digestion (Figure 2B lower panel, C). The disulfide peak was lost upon reduction (Figure 2B upper panel), yielding the corresponding thiol peptides. From this analysis we estimate that ~85% of C251 in the NPC1 N-terminal domain is disulfide bonded to C929 in NPC1's C-terminal domain. Figure 2 with 1 supplement see all Download asset Open asset 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 carbamidomethylated CQPPPPPMK from NPC1 P251C), orange traces represent m/z = 943.3462 (corresponding to carbamidomethylated NAAECDTY from NPC1 L929C), and green traces represent m/z = 626.6005 (corresponding to the disulfide formed between CQPPPPPMK and NAAECDTY). The peptide standards CQPPPPPMK and NAAECDTY were produced by solid phase synthesis and either reduced and carbamidomethylated or oxidized to the disulfide using Ellman’s reagent. (B) Extracted ion chromatograms from LC-MS analysis of proteolyzed NPC1. Colors as in (A). NPC1 protein was carbamidomethylated in the presence or absence of reducing agent prior to proteolysis. (C) EThcD mass spectrum from NPC1 sample in (B), demonstrating the reductive fragmentation of the putative disulfide precursor into ions with masses corresponding to the two constituent peptides. Peaks matching the mass of the peptide CQPPPPPMK (monoisotopic mass of thiol = 994.49 Da) are colored in purple; peaks matching the masses of the peptides NAAECDTY (monoisotopic mass of thiol radical = 885.32 Da) are colored in orange; peaks matching multiple charge states of the spectrum’s parent ions (m/z = 626.60, z = 3, corresponding to the disulfide) are colored in green. (D) MS1 mass spectra of NPC1 peptides whose disulfide content has been quantified using isotope-labeled iodoacetamide. Free thiols in purified NPC1 were labeled with 13C2D2-iodoacetamide (‘heavy’). Then the disulfides were reduced and the resulting reactive cysteines were labeled with iodoacetamide lacking isotope labels (‘light’) followed by proteolysis and LC-MS analysis. Control samples were labeled with the same reagent before and after reduction (‘light/reduce/light’ and ‘heavy/reduce/heavy’) to identify isotope distributions in the limiting cases. Colored peaks fall within 10 ppm of expected masses in the isotope envelope of carbamidomethylated CQPPPPPMK (monoisotopic m/z = 526.2569) or carbamidomethylated NAAECDTY (monoisotopic m/z = 943.3462); black peaks correspond to unrelated ions. Dashed gray lines indicate the expected m/z of the monoisotopic peak of the labeled peptides. To independently quantify the fraction of disulfide-bonded C251 and C929 residues, we labeled any free cysteines in NPC1 P251C/L929C/P249K/P259K with either 12C2H2 (‘light’) or 13C2D2 ('heavy’) iodoacetamide and monitored iodoacetamide labeling of NPC1 protein before and after chemical reduction. If the protein is fully disulfide-bonded, it should not incorporate 13C2D2 heavy iodoacetamide prior to reduction, enabling us to quantify precisely the fraction of NPC1 protein with a disulfide-protected cysteine. Samples are thus reacted with either light or heavy iodoacetamide, reduced, and then treated again with either light or heavy iodoacetamide. Control experiments using either light/light or heavy/heavy iodoacetamide in both the first and second rounds of labeling (Figure 2D, top panels) provided ‘standard’ spectra for the possible peptide products. When NPC1 P251C/L929C/P249K/P259K was first reacted with 13C2D2 heavy iodoacetamide, then reduced and subjected to another round of reaction with light iodoacetamide (Figure 2D, bottom row), NPC1 C251 and C929 were protected from heavy iodoacetamide labeling prior to chemical reduction—indicating they are disulfide-bonded. The two sites provided consistent measurements of the extent of disulfide bonding: measurement of peptide CQPPPPPMK indicated that 84% of P251C was protected from labeling by participation in a disulfide bond and 88% of the L929C site in the peptide NAAECDTY was protected. This extent of disulfide bonding is consistent with the detected functionality of the NPC1 P251C/L929C/P249K/P259K mutant protein and its correct localization to lysosomes (Figure 1F; Figure 1—figure supplement 1). All together, these data indicate that movement of the N-terminal domain away from the rest of the protein via the polyproline linker is not required for cholesterol export from lysosomes. Inter-domain mobility appears to be important for cholesterol transport Using photo-reactive, cross-linkable cholesterol, Hulce et al. (2013) identified cholesterol-binding peptides proteome-wide; their dataset included NPC1-derived peptides (highlighted in red, Figure 3A). The cholesterol-interacting peptides are located at the interface of the MLD and CTD as well as within the cytoplasmic loop connecting transmembrane domain TM7 to TM8. This 14-residue loop composed of residues 800–813 (broken line in Figure 3A) was not ordered in the high-resolution crystal structure of N-terminal domain-deleted NPC1 (PDBID: 5u74), and thus is likely mobile. We tested whether this loop is required for NPC1 function by deleting five residues (807-811) in mouse NPC1. The proper folding of this mutant was assessed by monitoring its intracellular localization in NPC1-/- HeLa cells (Figure 3B); co-localization with endogenous LAMP1 confirmed that this mutant NPC1 is correctly delivered to lysosomes. Figure 3 Download asset Open asset 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 (PDBID: 5u74). The disordered cytoplasmic loop residues 800–814 are shown as a blue dotted line. (B) Confocal immunofluorescence microscopy analysis of the localization of mouse NPC1-Δ807–811, NPC1-807-811Ala and LAMP1 proteins in HeLa cells (bar, 20 μm). White boxes in images indicate regions of cells enlarged in the insets shown at the lower right of each image. (C) Confocal immunofluorescence microscopy of cholesterol accumulation rescue. NPC1−/− HeLa cells were transfected with GFP-tagged mouse NPC1-Δ807–811 or mouse NPC1-807-811Ala plasmids for 48 h and assayed for cholesterol accumulation rescue as in Figure 1 (bar, 20 μm). (D) Quantitation of cholesterol accumulation rescue using flow cytometry. GFP-positive cells with similar expression levels were analyzed: 2480 NPC1; 427 NPC1-Δ807–811; 764 NPC1-807-811Ala; LAMP1 expressing control, 1753 cells counted. Shown are the normalized data from mean fluorescence intensity flow cytometry values. Despite its proper subcellular localization (Figure 3B), Δ807–811-mouse NPC1 could not rescue the cholesterol accumulation seen in NPC1-/- HeLa cells, as determined by immuno-fluorescence light microscopy (Figure 3C) as well as quantitative flow cytometry of cells expressing comparable amounts of the rescue construct (Figure 3D). These residues could contribute mobility for adjacent residues or specific interaction sequences needed for cholesterol export. It is worth noting that this TM7/TM8 loop lies adjacent to a binding site for cross-linkable cholesterol (Hulce et al., 2013). We replaced residues 807–811 with alanines, maintaining the length of the loop sequence. Co-localization with endogenous LAMP1 protein confirmed the proper subcellular localization of the mouse NPC1-807-811Ala mutant (Figure 3B) and similar to wild type NPC1, the 807-811Ala mutant protein fully rescued cholesterol accumulation in lysosomes of NPC1-/- transfected cells, monitored by immunofluorescence light microscopy (Figure 3C) and quantitative flow cytometry (Figure 3D). These data show that the cytoplasmic loop connecting TM7 and TM8 is indispensable for NPC1-mediated cholesterol export, and length is more important than specific amino acid sequences. These data imply that mobility of the protein helices is necessary for transport, with cytoplasmically oriented residues contributing in important ways. It is possible that this small truncation (Δ807–811) may strain the orientation of alpha helices in this region of the protein, but note that these five amino acids were among 14 residues that were not ordered in the high resolution crystal structure (PDBID: 5u74), and the truncation was designed with the goal of maintaining this structure. Nevertheless, the data do not rule out the possibility that this deletion simply favors a conformation of the protein that does not facilitate cholesterol transport. Cholesterol cross-linked NPC1 peptides also lie across the interface of the MLD and CTD (Figure 3A), suggesting that after binding to the N-terminal domain, cholesterol might traverse between interface residues. If so, locking these domains together with a precisely localized disulfide bond should block NPC1 function. To restrict the movement of the MLD and CTD with respect to one another, cysteines were introduced in place of A521 and K1013 to link these domains via a disulfide bond (Figure 4A). Co-localization of A521C/K1013C mouse NPC1 with endogenous LAMP1 confirmed the proper subcellular localization of this mutant in lysosomes (Figure 4B). The formation of this disulfide bond was also monitored by mass spectrometry; the A521C peptide APCSLNDTSLL was readily detected in reduced samples and recovered at 15% of the level in non-reduced samples, consistent with 85% disulfide bond formation (Figure 4—figure supplement 1). Figure 4 with 1 supplement see all Download asset Open asset 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 immunofluorescence microscopy analysis of mouse NPC1-A521C/K1013C and LAMP1 proteins in HeLa cells (bar, 20 μm). White boxes in images indicate regions of cells enlarged in the insets shown at the lower right of each image. (C) Confocal immunofluorescence microscopy of cholesterol accumulation rescue for NPC1-A521C or mouse NPC1-A521C/K1013C. (D) Flow cytometry of the rescue experiment analyzed in (C). GFP-positive cells with similar expression levels were analyzed: 17746 NPC1-/- cells; 1315 NPC1 wild type; 1137 NPC1-A521C/K1013C cells; 837 NPC1-A521C cells; cell numbers were normalized for comparison. Remarkably, when tested for its ability to rescue cholesterol accumulation, A521C/K1013C-NPC1 failed to rescue cholesterol accumulation in NPC1-/- transfected HeLa cells, monitored by immunofluorescence light microscopy (Figure 4C) and quantitative flow cytometry (Figure 4D). As an additional control, we also monitored the activity of an NPC1 protein in which only one cysteine mutation was introduced: NPC1-/- HeLa cells transfected with A521C-mouse NPC1 were fully rescued for cholesterol accumulation, monitored by light microscopy and flow cytometry (Figure 4C,D). Together, these data suggest that NPC1 only functions when the MLD and CTD can move in relation to one another. This essential mobility is consistent with a model in which cholesterol passes through the interface between the MLD and CTD as part of the cholesterol transport process. Molecular dynamics simulations of NPC1 and mutant proteins We sought additional hints to the mechanism of NPC1 cholesterol transport using molecular dynamics (MD) simulations. Conformational dynamics of wild type and the above-mentioned NPC1 protein constructs were analyzed by measuring the RMSD (root mean square deviation in Å) of protein backbone atoms, as sampled during the MD trajectories (Figure 5). NPC1 dynamics were characterized by a high degree of flexibility of the linker region (residues 247–266) connecting the N-terminal domain and helix 1 of the transmembrane domain, as well as a high degree of disordered secondary structure in the loops facing the cytoplasm (see Figure 1A). Notably, during our simulations, the N-terminal domain maintained its interface with the MLD and CTD and did not exhibit any large-scale hinging motion toward the transmembrane domains. The NPC1 mutant in which the MLD and CTD are locked together (A521C/K1013C) showed a similar range of motion for protein backbone atoms to that of wild type NPC1 (Figure 5). On the other hand, mutants in which the N-terminal domain was locked to the CTD (P251C/L929C) showed smaller RMS deviations relative to wild type. Locking the relatively disordered linker region of the N-terminal domain to the CTD removed a large source of protein flexibility. Similarly, the large RMSD values measured with mutant Δ807–811 stem primarily from increased disorder in the loops on the cytoplasmic side of the membrane (Figure 5). Figure 5 Download asset Open asset 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. To evaluate the extent of long-range concerted motion between protein domains, the distance correlation coefficients (DiCC) were calculated between the four domains (NTD, MLD, CTD, and the transmembrane domains; Table 1). For highly correlated motion between protein domains, the DiCC approach 1.00, whereas for uncorrelated motion the DiCC approach zero. In the wild type protein, MLD and the TMD showed the highest degree of correlated motion, whereas all mutants exhibit altered behavior. P251C/L929C showed the highest correlation between the transmembrane domains and CTD. In A521C/K1013C, the largest DiCC was calculated between the MLD and CTD (0.850), reflecting the concerted domain motion as a result of the disulfide bridge locking these domains together. Table 1 Distance correlation coefficients for mutants analyzed. Strongest inter-domain correlations are indicated in bold. A521C+K1013CNTDMLDCTDTMDNTD--------MLD--1.0000.8500.810CTD--0.8501.0000.594TMD--0.8100.5941.000P251C+L929CNTDMLDCTDTMDNTD1.0000.9690.9450.931MLD0.9691.0000.9190.942CTD0.9450.9191.0000.952TMD0.9310.9420.9521.000Δ807-811NTDMLDCTDTMDNTD1.0000.4960.5220.391MLD0.4961.0000.8360.398CTD0.5220.8361.0000.395TMD0.3910.3980.3951.000WTNTDMLDCTDTMDNTD1.0000.56904340.529MLD0.5691.0000.7510.811CTD0.4340.7511.0000.617TMD0.5290.8110.6171.000 Taken together, these results highlight the extent to which local changes in the NPC1 protein are propagated through the entire protein, affecting long-range domain motion. Moreover, the data support a model in which NPC1 protein relies on ‘cross-talk’ between domains, and these mechanisms are likely employed during sterol transfer. Inter-domain mobility is also important for cholesterol uptake by NPC1L1 Because NPC1L1 mediates cholesterol transport from the cell surface, we could test whether inter-domain flexibility is also important for this related cholesterol transporter using orthogonal assays. Figure 6A shows a model of NPC1L1 obtained by threading its sequence onto the structure of NPC1 using Swiss-Model (PDB: 5u74; Waterhouse et al., 2018). NPC1L1 mutants that were constrained at their cytoplasmic loop (Δ820–824) or restricted in terms of the mobility of the MLD in relation to the CTD (F532C/I1022C) were designed in a manner analogous to the NPC1 mutants; these failed to import cholesterol into HEK293T cells expressing these proteins at the cell surface (Figure 6B). As shown previously (Zhang et al., 2011), NPC1L1 missing its N-terminal domain also failed to import cholesterol efficiently, although a low level of uptake was observed when compared with control samples (Figure 6B). Lack of transport was not a result of differences in cell surface localization or protein levels, as we determined the localizations and amounts of all the mutant proteins using a cell surface biotinylation assay in conjunction with immunoblotting (Figure 6C and Johnson and Pfeffer, 2016). These experiments confirm the importance of interdomain flexibility for cholesterol transport by both NPC1 and NPC1L1 proteins." @default.
• W4248892569 created "2022-05-12" @default.
• W4248892569 date "2020-04-20" @default.
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• W4248892569 title "Decision letter: Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins" @default.
• W4248892569 doi "https://doi.org/10.7554/elife.57089.sa1" @default.
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