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).

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.

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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.

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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

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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).

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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

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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

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

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.

Figure 6

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The MLD plays a unique role in NPC1L1

We have shown previously that NPC1’s MLD binds NPC2 to facilitate transfer of cholesterol from NPC2 onto NPC1’s N-terminal domain (Deffieu and Pfeffer, 2011; Li et al., 2016b). Unlike NPC1, NPC1L1 does not receive cholesterol from an NPC2-like protein in the intestine—instead, it receives cholesterol from bile salt micelles (Yamanashi et al., 2007; Haikal et al., 2008). We were intrigued by the possibility that the MLD of NPC1L1 might play an analogous role to its related domain in NPC1 and that it might bind bile salt micelles to facilitate transfer of cholesterol onto NPC1L1’s N-terminal domain.

To test this, we established a binding assay using purified, soluble NPC1L1 N-terminal domain or NPC1L1 MLD. ³H-cholesterol was presented to these proteins either mixed with Nonidet P40 (NP-40) below its critical micelle concentration (CMC), according to established methods used to measure cholesterol binding to NPC1 and NPC1L1 NTD (Infante et al., 2008; Kwon et al., 2011), or in the form of a bile salt micelle (Figure 7; Figure 7—figure supplement 1).

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As expected, purified NPC1L1 N-terminal domain bound cholesterol when it was presented in the presence of sub-CMC NP-40 (Kwon et al., 2011; Figure 7 top); under these conditions, no cholesterol binding was detected to purified NPC1L1 MLD. In contrast, when cholesterol was presented to the N-terminal domain in the form of a bile salt micelle, no binding was detected, but significant binding was observed for reactions containing NPC1L1 MLD (Figure 7, bottom). These data indicate that NPC1L1 MLD contains a specific binding site for cholesterol in the form of a bile salt micelle.

Binding of cholesterol in the form of a bile salt micelle to the MLD was not altered by addition of up to 100 µM of the NPC1L1 inhibitor, ezetimibe, which relies on NPC1L1 MLD residues for high-affinity binding (Weinglass et al., 2008). These experiments reveal, for the first time, that MLD may function to bind bile salt micelles and thus facilitate capture of cholesterol by the N-terminal domain as part of the cholesterol uptake process. In addition, they suggest that ezetimibe inhibition of NPC1L1 protein is likely not a result of interference with bile salt micelle binding to MLD.

Domain interface-locking contributes to the mechanism of ezetimibe inhibition

The mechanisms by which small molecules inhibit transporter proteins can provide important clues to the mechanisms by which transporters normally function. As mentioned earlier, sequences located within NPC1L1 MLD are important for high-affinity ezetimibe binding (Weinglass et al., 2008). We determined the possible additional contributions of other NPC1L1 domains to ezetimibe binding. As shown in Figure 8A, cells expressing wild type NPC1L1 showed binding to ³H-ezetimibe that was specific as it could be competed away by addition of excess unlabeled ezetimibe. Expression of an N-terminal domain-deleted NPC1L1 protein abolished ezetimibe binding, despite the efficient delivery of ΔN-NPC1L1 to the plasma membrane of transfected HEK293T cells; ~30% of both wild type and mutant NPC1L1 proteins were localized to the cell surface (Figure 8B). Thus, NPC1L1 N-terminal domain sequences are also important for ezetimibe binding.

Figure 8

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It is important to note that the MLD residues previously identified as being important for ezetimibe binding by Weinglass et al. (2008) are predicted to lie at the interface between the MLD and CTD in the structure model. Given the apparent importance of inter-domain interfaces in cholesterol transport by NPC1, we tested the possibility that inter-domain interface residues create a binding site for ezetimibe on NPC1L1. Small molecule binding adjacent to interface residues would have the same consequence as the disulfide-bonded, locked mutants—it could restrain protein dynamics and therefore block cholesterol transport.

Figure 9A shows the effect of mutating a number of residues that are predicted to be important for contacts between NPC1L1 N-terminal domain and the MLD or CTD. Table 2 summarizes the rationale for each of the mutations generated. Remarkably, single point mutations of representative residues from each of these interfaces abolished the ability of ezetimibe to bind NPC1L1 (Figure 9A). Once again, we verified the cell surface localization of many of these mutant proteins; in each case, about 30% of the protein was present at the cell surface (Figure 9B). These data provide new information about the structural basis for ezetimibe interaction with NPC1L1 protein and support a model whereby ezetimibe blocks NPC1L1 by locking the various domains together, potentially plugging a potential cholesterol channel through the molecule (Winkler et al., 2019; Long et al., 2020).

Figure 9

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Table 2

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)

We have shown here that dynamic, inter-domain interactions within NPC1 and NPC1L1 are essential for the ability of these proteins to mediate cholesterol transport out of lysosomes or across the plasma membrane, respectively. Specifically, mutants that can lock the MLD to the CTD at the interface of these domains interfere with cholesterol transport; residues that comprise a loop at the cytoplasmic face of the transmembrane domains are needed to provide inter-domain mobility and/or an unconstrained conformation, and can be replaced with polyalanine for full function. Moreover, we have shown that ezetimibe inhibition of NPC1L1 relies on its interaction with multiple domains and depends on sequences that are predicted to contribute to domain interface interactions in the protein structure. A highly plausible model for ezetimibe inhibition would be that it functions in a manner analogous to the disulfide bond mutants: locking the domains to one another and thereby blocking cholesterol transport. By binding at the interface, ezetimibe could also plug a channel between these domains. We showed for the first time that purified NPC1L1 MLD binds bile salt micelles. Although we cannot exclude the possibility that ezetimibe blocks bile salt interaction in full-length NPC1L1, the mutagenesis experiments support the importance of interface residues for drug binding, which will have the additional consequence of blocking inter-domain dynamics.

It is currently accepted that NPC2 transfers bound cholesterol to the NPC1 N-terminal domain; however, the subsequent steps by which cholesterol transporters move cholesterol across the membrane remain unclear. Determination of the structure of NPC1 revealed that the N-terminal domain cholesterol binding site is located on the opposite side of the protein to the sterol-sensing domain; this finding led to the proposal that the N-terminal domain may transfer its cholesterol to an adjacent NPC1 molecule (Li et al., 2016a). The experiments presented here do not address this possibility directly, but they demonstrate that cholesterol transport does not require full release of the N-terminal domain from the rest of the protein. Locking the polyproline linker that attaches the N-terminal domain to the rest of NPC1 protein had no impact on the ability of NPC1 to export cholesterol from lysosomes using kinetically sensitive and quantitative assays for this process. These results suggest that the N-terminal domain need not hinge away from the rest of the protein to transfer cholesterol from the N-terminal domain to other parts of the protein and/or across the membrane. Note that the portion of the linker most proximal to the NTD will nevertheless retain some flexibility, despite the presence of the additional disulfide bond (Figure 1B).

Taken together, our data support a model in which cholesterol can pass directly through NPC1, either to the sterol-sensing domain or all the way through the membrane bilayer. This model is further supported by recent computations and structural experiments predicting a cholesterol transfer path through the center of NPC1 (Elghobashi-Meinhardt, 2019; see also Wheeler et al., 2019; Winkler et al., 2019; Long et al., 2020). Because of the importance of cytoplasmically oriented residues for NPC1 function, we favor a model in which the sterol-sensing domain binding site represents a regulatory site and is not necessarily involved in the transport process per se.

Precedent for cholesterol passing through a related transporter comes from the structural work of Nieng Yan and Xiaochun Li and their colleagues who have recently reported structures of the related Patched (PTCH) protein, which is important for Hedgehog signaling (Gong et al., 2018; Qi et al., 2018a; Qi et al., 2018b). PTCH has 12 transmembrane domains instead of 13; it also lacks the cholesterol binding N-terminal domain of NPC1 and NPC1L1. Transmembrane domains 2–6 comprise a sterol-sensing domain analogous to transmembrane domains 3–7 of NPC1. Yan and colleagues detected two sterol molecules in the PTCH structure: one in a cavity located between the two extracellular domains and the second, adjacent to the sterol-sensing domain (Gong et al., 2018). Their mutant analysis suggests that cholesterol binding causes significant conformational changes, with an untwisting of the interactions between the extracellular domains and reorientation of certain TMs. Qi et al. (2018b) found two sterol-like ligand densities in the transmembrane domain of each PTCH molecule, one in the sterol-sensing domain and the other near transmembrane domain 12. Importantly, the structural analysis is consistent with a tunnel in the PTCH protein, through which a sterol could pass.

Does NPC1 also bind cholesterol in a cavity between its extracellular domains, analogous to PTCH? Does NPC1 contain a similar channel through which cholesterol might pass? Important hints came initially from Cravatt and colleagues (Hulce et al., 2013), whose proteome-wide analysis of cholesterol binding proteins revealed novel cholesterol binding locations in NPC1 (Figure 3A). Our mutant protein functional analysis is consistent with the possibility that NPC1 and NPC1L1 undergo significant conformational transitions to accomplish transport of cholesterol across the membrane.

While this study was under review, Xiaochun Li's team reported the cryo-EM structure of NPC1 with an added inhibitor, itraconazole, occupying a central cleft at the top of NPC1's MLD and CTD (Long et al., 2020). This finding meshes well with our proposal for how ezetimibe blocks cholesterol transport by NPC1L1 at a similar location in the corresponding structure. Whether cholesterol exits this channel at the sterol-sensing domain (see also Winkler et al., 2019) or proceeds through the molecule to the other side of the bilayer is not yet clear, but our finding that constriction of the proteins' cytoplasmic domains blocks function suggests that the channel may indeed pass all the way through the transmembrane domain. Structural analysis of such mutant proteins may help to resolve this question.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Piyali Saha

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Present address

   Merck Research Laboratories, South San Francisco, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   is affiliated with Merck Research Laboratories. The author has no financial interests to declare. This work was completed prior to her employment at Merck and this basic study will not impact any sales of Zetia, the tradename of Ezetimibe.

2. Justin L Shumate

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

3. Jenna G Caldwell

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6392-4794

4. Nadia Elghobashi-Meinhardt

   Department of Chemistry, Technische Universität Berlin, Berlin, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1023-6856

5. Albert Lu

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

6. Lichao Zhang

   Chan Zuckerberg BioHub, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1811-7568

7. Niclas E Olsson

   Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, United States

   Present address

   Calico Life Sciences LLC, South San Francisco, United States

   Contribution

   Resources, Data curation, Formal analysis

   Competing interests

   is affiliated with Calico Life Sciences LLC. The author has no financial interests to declare.

   "This ORCID iD identifies the author of this article:" 0000-0002-9746-0542

8. Joshua E Elias

   Chan Zuckerberg BioHub, Stanford, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision

   Competing interests

   No competing interests declared

9. Suzanne R Pfeffer

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   pfeffer@stanford.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

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