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• W2973505555 abstract "•The yeast NPC system is a model system for sterol membrane integration in eukaryotes•Structures of NCR1 and NPC2 suggest a mechanism for sterol trafficking and integration•Our data support a tunnel pathway between the MLD and CTD to bypass the glycocalyx•An acidic pseudo-symmetry amino acid pair in NCR1 suggests proton-driven sterol translocation Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C. Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C. Sterols are an essential component of membranes in all eukaryotic cells. In humans, Niemann-Pick type C proteins NPC1 and NPC2 bind cholesterol and are essential for lysosomal membrane integration from where cholesterol is redistributed to other cellular membranes (Carstea et al., 1997Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. et al.Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Google Scholar, Loftus et al., 2002Loftus S.K. Erickson R.P. Walkley S.U. Bryant M.A. Incao A. Heidenreich R.A. Pavan W.J. Rescue of neurodegeneration in Niemann-Pick C mice by a prion-promoter-driven Npc1 cDNA transgene.Hum. Mol. Genet. 2002; 11: 3107-3114Google Scholar, Naureckiene et al., 2000Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Google Scholar). Disturbance of this pathway leads to Niemann-Pick disease type C, a neurodegenerative disease where cholesterol and other lipids accumulate in the lysosomes (Pentchev, 2004Pentchev P.G. Niemann-Pick C research from mouse to gene.Biochim. Biophys. Acta. 2004; 1685: 3-7Google Scholar). Furthermore, NPC1 has been identified as a key virulence factor for the Filoviruses Ebola and Marburg (Carette et al., 2011Carette J.E. Raaben M. Wong A.C. Herbert A.S. Obernosterer G. Mulherkar N. Kuehne A.I. Kranzusch P.J. Griffin A.M. Ruthel G. et al.Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.Nature. 2011; 477: 340-343Google Scholar, Côté et al., 2011Côté M. Misasi J. Ren T. Bruchez A. Lee K. Filone C.M. Hensley L. Li Q. Ory D. Chandran K. Cunningham J. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection.Nature. 2011; 477: 344-348Google Scholar). The molecular mechanism of sterol membrane integration is still unclear, despite having been studied extensively. NPC2, a soluble intra-lysosomal protein, binds sterols with distinct polarity (polar head group exposed and aliphatic tail buried in the cavity) and is able to hand off this sterol to the luminal N-terminal domain (NTD) of the lysosomal membrane protein NPC1 with reversed polarity (polar head group buried in the pocket) (Deffieu and Pfeffer, 2011Deffieu M.S. Pfeffer S.R. Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding.Proc. Natl. Acad. Sci. USA. 2011; 108: 18932-18936Google Scholar, Friedland et al., 2003Friedland N. Liou H.-L. Lobel P. Stock A.M. Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease.Proc. Natl. Acad. Sci. USA. 2003; 100: 2512-2517Google Scholar, Kwon et al., 2009Kwon H.J. Abi-Mosleh L. Wang M.L. Deisenhofer J. Goldstein J.L. Brown M.S. Infante R.E. Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.Cell. 2009; 137: 1213-1224Google Scholar, Wang et al., 2010Wang M.L. Motamed M. Infante R.E. Abi-Mosleh L. Kwon H.J. Brown M.S. Goldstein J.L. Identification of surface residues on Niemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 in lysosomes.Cell Metab. 2010; 12: 166-173Google Scholar, Xu et al., 2007Xu S. Benoff B. Liou H.-L. Lobel P. Stock A.M. Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease.J. Biol. Chem. 2007; 282: 23525-23531Google Scholar). Through an unknown mechanism, the sterol bound to the NTD is then believed to be passed to the lysosomal membrane for integration (Davies and Ioannou, 2000Davies J.P. Ioannou Y.A. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.J. Biol. Chem. 2000; 275: 24367-24374Google Scholar, Davies et al., 2000Davies J.P. Chen F.W. Ioannou Y.A. Transmembrane molecular pump activity of Niemann-Pick C1 protein.Science. 2000; 290: 2295-2298Google Scholar). Several sterol binding sites have been suggested in NPC1, and sterol integration into the membrane involves a membrane-buried sterol sensing domain (SSD), but the structural and functional relationship between these sites in NPC proteins remains unknown (Lu et al., 2015Lu F. Liang Q. Abi-Mosleh L. Das A. De Brabander J.K. Goldstein J.L. Brown M.S. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.eLife. 2015; 4 (Published online December 8, 2015)https://doi.org/10.7554/eLife.12177Google Scholar, Ohgami et al., 2004Ohgami N. Ko D.C. Thomas M. Scott M.P. Chang C.C.Y. Chang T.-Y. Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain.Proc. Natl. Acad. Sci. USA. 2004; 101: 12473-12478Google Scholar, Ohgane et al., 2013Ohgane K. Karaki F. Dodo K. Hashimoto Y. Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.Chem. Biol. 2013; 20: 391-402Google Scholar). The luminal side of the lysosomal membrane is covered in an ∼60 Å thick glycocalyx, a polysaccharide matrix coating that preserves the integrity of the lysosomal membrane protecting the cytosol from degradative enzymes inside the lysosome lumen (Neiss, 1984Neiss W.F. A coat of glycoconjugates on the inner surface of the lysosomal membrane in the rat kidney.Histochemistry. 1984; 80: 603-608Google Scholar). It has been speculated that NPC1 could help sterols bypass this protective layer (Gong et al., 2016Gong X. Qian H. Zhou X. Wu J. Wan T. Cao P. Huang W. Zhao X. Wang X. Wang P. et al.Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection.Cell. 2016; 165: 1467-1478Google Scholar, Li et al., 2016aLi X. Wang J. Coutavas E. Shi H. Hao Q. Blobel G. Structure of human Niemann-Pick C1 protein.Proc Natl Acad Sci USA. 2016; 113: 8212-8217Google Scholar, Pfeffer, 2019Pfeffer S.R. NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes.J. Biol. Chem. 2019; 294: 1706-1709Google Scholar). In yeast, the degradation process of the lysosome is delegated to the vacuole (Schulze et al., 2009Schulze H. Kolter T. Sandhoff K. Principles of lysosomal membrane degradation: Cellular topology and biochemistry of lysosomal lipid degradation.Biochim. Biophys. Acta. 2009; 1793: 674-683Google Scholar). The S. cerevisiae NPC system, constituting NCR1 and NPC2 and located in the yeast vacuole, has been studied extensively to obtain insight into the function and dynamics of NPC proteins, and both NPC1- and NPC2-deficient mammalian cells can be rescued by the S. cerevisiae counterpart (Berger et al., 2005aBerger A.C. Hanson P.K. Wylie Nichols J. Corbett A.H. A yeast model system for functional analysis of the Niemann-Pick type C protein 1 homolog, Ncr1p.Traffic. 2005; 6: 907-917Google Scholar, Berger et al., 2005bBerger A.C. Vanderford T.H. Gernert K.M. Nichols J.W. Faundez V. Corbett A.H. Saccharomyces cerevisiae Npc2p is a functionally conserved homologue of the human Niemann-Pick disease type C 2 protein, hNPC2.Eukaryot. Cell. 2005; 4: 1851-1862Google Scholar, Jacquier and Schneiter, 2012Jacquier N. Schneiter R. Mechanisms of sterol uptake and transport in yeast.J. Steroid Biochem. Mol. Biol. 2012; 129: 70-78Google Scholar, Malathi et al., 2004Malathi K. Higaki K. Tinkelenberg A.H. Balderes D.A. Almanzar-Paramio D. Wilcox L.J. Erdeniz N. Redican F. Padamsee M. Liu Y. et al.Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution.J. Cell Biol. 2004; 164: 547-556Google Scholar, Munkacsi et al., 2011Munkacsi A.B. Chen F.W. Brinkman M.A. Higaki K. Gutiérrez G.D. Chaudhari J. Layer J.V. Tong A. Bard M. Boone C. et al.An “exacerbate-reverse” strategy in yeast identifies histone deacetylase inhibition as a correction for cholesterol and sphingolipid transport defects in human Niemann-Pick type C disease.J. Biol. Chem. 2011; 286: 23842-23851Google Scholar, Tsuji et al., 2017Tsuji T. Fujimoto M. Tatematsu T. Cheng J. Orii M. Takatori S. Fujimoto T. Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole.eLife. 2017; 6 (Published online June 7, 2017)https://doi.org/10.7554/eLife.25960Google Scholar). The yeast NPC system (NCR1 and NPC2) shares 32% and 22% sequence identity with the human system (hNPC1 and hNPC2, respectively), but there is no structural framework to relate NCR1 and NPC2 to their human counterparts at the molecular level (Figures S1 and S2A).Figure S2Sequence Alignment and Crystal Structures of S. cerevisiae NPC2, Related to Figure 1Show full caption(A) Alignment between NPC2 (UniProt: Q12408), and hNPC2 (UniProt: P61916), based on a multiple sequence alignment in Promals3D followed by small manual adjustments based on structural comparison. Conserved residues are highlighted with black. Residues that could not be modeled in the structure have a gray font. Colored tubes and arrows represent alpha-helices and beta-strands. Signal peptide and pro-peptide are highlighted with gray boxes. Asparagines with observed glycosylation are highlighted with green and cysteines involved in disulfide bridges are highlighted with orange.(B) SDS-PAGE gel of the final sample for crystallization. Crystals of sterol-free NPC2. The black scale bar is 100 μm. Weighted 2FoFc density of the asymmetric unit, showing the threefold symmetry of the trimer contoured at 1.2 sigma.(C) Interaction between propeptide (purple) from symmetry related monomer to chain C (light orange) in sterol-free NPC2. Chain A and B does not have a similar interaction, and their propeptide cannot be resolved in the density.(D) Crystals of sterol-bound NPC2. The black scale bar is 100 μm. Weighted 2FoFc density of the trimer formed by chain A, B and C contoured at 1.2 sigma.(E) Asymmetric unit of sterol-bound NPC2 crystals containing 9 monomers, connected as 3 trimers (A-C, D-F and G-I). Five monomers have ergosterol bound (purple). The remaining four have density in the cavity that cannot be confidently modeled.(F) Weighted FoFc density (green) with ergosterol (purple) omitted shown at 3 sigma. To the right is shown a Polder map (green) at 6.0 sigma with CC(with ligand, input) = 0.84 and CC(without ligand, input) = 0.56. The difference in polder-map CC support a model with ligand included.(G) Weighted FoFc density (3.0 sigma) in the cavity of chain A and chain H of sterol bound NPC2. The sterol of chain A was omitted before refinement and map calculation. Density in chain A show a single sterol molecule, while the density in chain H could not be confidently modeled.(H) Overlay of human (dark gray) and bovine (white) NPC2 with yeast sterol bound NPC2 (rainbow). Ergosterol from the NPC2 structure is shown (purple) together with cholesterol sulfate (white and dark gray) from the human and bovine structures.(I) There is space enough for an additional sterol in the yeast NPC2 cavity as illustrated by in silico modeling of an additional ergosterol into the pocket (white).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Alignment between NPC2 (UniProt: Q12408), and hNPC2 (UniProt: P61916), based on a multiple sequence alignment in Promals3D followed by small manual adjustments based on structural comparison. Conserved residues are highlighted with black. Residues that could not be modeled in the structure have a gray font. Colored tubes and arrows represent alpha-helices and beta-strands. Signal peptide and pro-peptide are highlighted with gray boxes. Asparagines with observed glycosylation are highlighted with green and cysteines involved in disulfide bridges are highlighted with orange. (B) SDS-PAGE gel of the final sample for crystallization. Crystals of sterol-free NPC2. The black scale bar is 100 μm. Weighted 2FoFc density of the asymmetric unit, showing the threefold symmetry of the trimer contoured at 1.2 sigma. (C) Interaction between propeptide (purple) from symmetry related monomer to chain C (light orange) in sterol-free NPC2. Chain A and B does not have a similar interaction, and their propeptide cannot be resolved in the density. (D) Crystals of sterol-bound NPC2. The black scale bar is 100 μm. Weighted 2FoFc density of the trimer formed by chain A, B and C contoured at 1.2 sigma. (E) Asymmetric unit of sterol-bound NPC2 crystals containing 9 monomers, connected as 3 trimers (A-C, D-F and G-I). Five monomers have ergosterol bound (purple). The remaining four have density in the cavity that cannot be confidently modeled. (F) Weighted FoFc density (green) with ergosterol (purple) omitted shown at 3 sigma. To the right is shown a Polder map (green) at 6.0 sigma with CC(with ligand, input) = 0.84 and CC(without ligand, input) = 0.56. The difference in polder-map CC support a model with ligand included. (G) Weighted FoFc density (3.0 sigma) in the cavity of chain A and chain H of sterol bound NPC2. The sterol of chain A was omitted before refinement and map calculation. Density in chain A show a single sterol molecule, while the density in chain H could not be confidently modeled. (H) Overlay of human (dark gray) and bovine (white) NPC2 with yeast sterol bound NPC2 (rainbow). Ergosterol from the NPC2 structure is shown (purple) together with cholesterol sulfate (white and dark gray) from the human and bovine structures. (I) There is space enough for an additional sterol in the yeast NPC2 cavity as illustrated by in silico modeling of an additional ergosterol into the pocket (white). Clues to the mechanism of sterol uptake can be found in the cryo-electron microscopy (cryo-EM) structure of the full-length hNPC1 (Gong et al., 2016Gong X. Qian H. Zhou X. Wu J. Wan T. Cao P. Huang W. Zhao X. Wang X. Wang P. et al.Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection.Cell. 2016; 165: 1467-1478Google Scholar), the crystal structure of a large fragment of hNPC1 without the NTD (Li et al., 2016aLi X. Wang J. Coutavas E. Shi H. Hao Q. Blobel G. Structure of human Niemann-Pick C1 protein.Proc Natl Acad Sci USA. 2016; 113: 8212-8217Google Scholar), and the crystal structure of the middle luminal domain (MLD) of hNPC1 bound to hNPC2 (Li et al., 2016bLi X. Saha P. Li J. Blobel G. Pfeffer S.R. Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2.Proc. Natl. Acad. Sci. USA. 2016; 113: 10079-10084Google Scholar). Furthermore, recent breakthroughs in the related Patched protein family belonging to the same Resistance-Nodulation-Division (RND) superfamily as NPC1 proteins also provide hints toward a possible common core mechanism, since Patched is involved in a reverse process of sterol extraction from the membrane (Gong et al., 2018Gong X. Qian H. Cao P. Zhao X. Zhou Q. Lei J. Yan N. Structural basis for the recognition of Sonic Hedgehog by human Patched1.Science. 2018; 361 (Published online August 10, 2018)https://doi.org/10.1126/science.aas8935Google Scholar, Qi et al., 2018aQi X. Schmiege P. Coutavas E. Wang J. Li X. Structures of human Patched and its complex with native palmitoylated sonic hedgehog.Nature. 2018; 560: 128-132Google Scholar, Qi et al., 2018bQi X. Schmiege P. Coutavas E. Li X. Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex.Science. 2018; 362 (Published online October 5, 2018)https://doi.org/10.1126/science.aas8843Google Scholar, Zhang et al., 2018Zhang Y. Bulkley D.P. Xin Y. Roberts K.J. Asarnow D.E. Sharma A. Myers B.R. Cho W. Cheng Y. Beachy P.A. Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.Cell. 2018; 175: 1352-1364.e14Google Scholar). Despite this, we do not have a clear understanding of how NPC1 and NPC2 mediate transfer of sterols into the lysosomal membrane, and several different models currently co-exist (Li et al., 2016aLi X. Wang J. Coutavas E. Shi H. Hao Q. Blobel G. Structure of human Niemann-Pick C1 protein.Proc Natl Acad Sci USA. 2016; 113: 8212-8217Google Scholar, Pfeffer, 2019Pfeffer S.R. NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes.J. Biol. Chem. 2019; 294: 1706-1709Google Scholar, Trinh et al., 2018Trinh M.N. Brown M.S. Seemann J. Goldstein J.L. Lu F. Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1.eLife. 2018; 7 (Published online July 26, 2018)https://doi.org/10.7554/eLife.38564Google Scholar). Here, we present four structures of yeast NCR1 and NPC2. The crystal structure of NPC2 is solved in both a sterol-free and sterol-bound state. We measure sterol binding affinities and demonstrate that sterol transfer is possible between NPC2 and the NTD of NCR1. We show that one physiological function of NPC2 and NCR1 is to deliver sterols to the vacuole in living yeast cells, in analogy to the human system. The structures of NPC2 rationalize how sterols are captured and shuttled in the yeast vacuole, and they display an expanded binding site compared to its human counterpart indicative of a broader substrate range. We also present the first crystal structure, as well as the first single-particle cryo-EM structure, of NCR1. Both structures adopt a novel conformation not observed in the human NPC1. We identify a tunnel through the central core of NCR1 that links the NTD with the SSD and in which we observe a bound sterol. Based on our data, we propose a mechanism for sterol transport where NCR1 can bypass the glycocalyx via a tunnel to mediate sterol insertion into the membrane. The model explains puzzling results in the field related to the number of sterol binding sites, and it offers a rationale for why the sterol polarity switch from NPC2 to NTD is necessary for proper membrane integration in vivo. Our data suggest that the molecular mechanism of sterol transfer and sterol membrane integration is conserved from fungi to humans and offers a rationale for missense mutations found in patients with Niemann-Pick disease type C. We expressed, purified, and crystallized S. cerevisiae NPC2 (Figure S2B; see also Table S1). The sterol-free form of NPC2 is determined by experimental phasing to 2.8 Å (Rfree 26.2%) with 3 identical monomers (root-mean-square deviation, RMSD(CA) < 0.33 Å) in the asymmetric unit forming a trimer with 120 degree rotational symmetry. In the crystal packing, a propeptide (residue 24–34) from one monomer of the trimer extends to fold over a neighboring monomer from another trimer (Figure S2C). The NPC2 monomer adopts an open Ig-like beta sandwich fold with seven anti-parallel beta strands (Figure 1A). Strand 1, 2, and 5 form one side of the sandwich, while strand 3, 4, 6, and 7 form the other. A small alpha helix between strand 4 and 5 forms a cover (residues 109–123) enclosing part of a deep and elongated hydrophobic cavity (1,019 Å3) between the two sides of the sandwich, leaving space for substrate entry and exit at only one end of the sandwich. Two disulfide bonds and a single N-linked glycosylation stabilize the structure. The structure of NPC2 in complex with sterols was determined to 2.9 Å (Rfree 25.7%) (Figures 1B and S2D; see also Table S1). The asymmetric unit contains 9 monomers forming 3 trimers similar to the trimer in the sterol-free structure, but without the propeptide interaction between trimers (Figure S2E). A single ergosterol molecule is found in the hydrophobic binding cavity in 5 of the monomers, while the remaining 4 monomers have strong density features in the binding cavity that cannot be confidently classified but appear to be bulkier than ergosterol and may be other lipids (Figure S2F and S2G). The sterol polarity within the cavity is such that the polar head group points out and the aliphatic tail into the cavity in the same orientation as observed in previous human and bovine NPC2 structures (Figure S2H) (Li et al., 2016bLi X. Saha P. Li J. Blobel G. Pfeffer S.R. Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2.Proc. Natl. Acad. Sci. USA. 2016; 113: 10079-10084Google Scholar, Xu et al., 2007Xu S. Benoff B. Liou H.-L. Lobel P. Stock A.M. Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease.J. Biol. Chem. 2007; 282: 23525-23531Google Scholar). The sterol resides in a hydrophobic cavity lined by the side chains of Pro67, Leu73, Val95, Leu102, Leu103, Ile138, Val142, Tyr147, Val149, Val168, and Phe170, some of which form non-specific hydrophobic interactions between NPC2 and the sterol (Figure 1C). The cavity is of sufficient size that two sterols could fit side by side in the opening, but such a setup is not supported by the observed density (Figures S2G and S2I). There is no difference in conformation of sterol-free and sterol-bound NPC2 (RMSD(CA) < 0.3 Å) (Figure 1B), similar to the bovine sterol-free versus sterol-bound NPC2 (Xu et al., 2007Xu S. Benoff B. Liou H.-L. Lobel P. Stock A.M. Structural basis of sterol binding by NPC2, a lysosomal protein deficient in Niemann-Pick type C2 disease.J. Biol. Chem. 2007; 282: 23525-23531Google Scholar). While yeast NPC2 has a similar sandwich fold and almost identical binding of a single sterol as human and bovine NPC2, the global structure displays significant differences (e.g., to bovine sterol-bound form RMSD(CA) 3.0 Å) (Figure S2H). NPC2 has a much larger binding cavity compared to human and bovine NPC2 because it lacks a disulfide bond that in the human and bovine NPC2 pushes the beta sandwich together opposite the sterol binding site. Furthermore, multiple bulky side chains are facing the cavity in both human and bovine NPC2 but not in yeast NPC2, reducing the size of these cavities (e.g., bovine NPC2 cavity: 186 Å3) (Figures 1D). Despite this, the observed sterol binding site in all three is very similar with respect to position and orientation of the sterol, with a small difference being a slight tilt of the sterol in yeast NPC2 compared to bovine and human NPC2 (Figure 1E). It has previously been shown that yeast NPC2 can rescue human NPC2-deficient fibroblasts (Berger et al., 2005bBerger A.C. Vanderford T.H. Gernert K.M. Nichols J.W. Faundez V. Corbett A.H. Saccharomyces cerevisiae Npc2p is a functionally conserved homologue of the human Niemann-Pick disease type C 2 protein, hNPC2.Eukaryot. Cell. 2005; 4: 1851-1862Google Scholar), and we can now rationalize this, as essential components of sterol binding are conserved between yeast NPC2 and human NPC2. To establish the binding properties of the yeast NPC system, we assayed binding affinities of NPC2 and the NTD of NCR1 using two different approaches: (1) fluorescence-detected resonance energy transfer (FRET) from aromatic residues to an intrinsically fluorescent ergosterol analog, dehydroergosterol (DHE), and (2) binding of radiolabeled cholesterol. When exciting NPC2 at 280 nm, sterol binding can be detected as fluorescence of DHE emitting between 360 and 400 nm with three characteristic peaks (Figure S3A). This FRET effect from NPC2’s Tyr and Phe shows that DHE binds to NPC2 with nanomolar affinity (Kd = 121 nM) (Figure 2A). Using radiolabeled cholesterol in a different experimental setup, we observe a binding affinity in the same nanomolar range (Kd = 591 nM) (Figure 2B). Binding competition assays show that the antifungal lipid edelfosine interferes strongly with cholesterol binding, while the hNPC1 inhibitor U18666A and the cholesterol trafficking inhibitor Cepharanthine inhibit binding more weakly (Figure 2C). In combination with the large binding cavity observed in the crystal structure, these data suggest that yeast NPC2 could be involved in general hydrophobic substrate trafficking, but this needs to be investigated further in order to identify which substrates it shuttles.Figure 2Sterol Affinity of NPC2 and NTD and Sterol Transfer AssaysShow full caption(A) FRET signal shows NPC2 binds dehydroergosterol (DHE) with a Kd = 121 ± 75 nM. Data points show mean ± SEM of four (n = 4) independent experiments.(B) NPC2 binds radioactive cholesterol with a Kd = 591 ± 123 nM. Data points show mean ± SD of four (n = 4) independent experiments.(C) NPC2 competition assay. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 by Student’s t test. Data for all radiolabeled binding assays are mean ± SD of five (n = 5) independent experiments.(D) FRET signal shows NTD binds dehydroergosterol (DHE) with a Kd = 483 ± 83 nM. Data points show mean ± SEM of three (n = 3) independent experiments.(E) NTD binds radioactive cholesterol with a Kd = 675 ± 210 nM. Data points show mean ± SD of four (n = 4) independent experiments.(F) NTD competition assay. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; and ∗∗∗∗p ≤ 0.0001 by Student’s t test. Data for all radiolabeled binding assays are mean ± SD of five (n = 5) independent experiments.(G) FRET-based transfer assay of DHE from NTD to NPC2 (full line) and from NPC2 to NTD (dashed line). Data points show mean ± SEM of three (n = 3) independent experiments.(H) Transfer assay with his-tagged NPC2 as donor and untagged NTD (dotted line) or untagged NPC2 (full line) as acceptor. Data points show mean ± SD of five (n = 5) independent experiments.(I) Transfer assay with his-tagged NTD as donor and untagged NTD (full line) or untagged NPC2 (dotted line) as acceptor. Data points show mean ± SD of five (n = 5) independent experiments.(J) Transfer assay with his-tagged NPC2 as donor and wild-type or mutated NCR1 as acceptor. Data points show mean ± SD of three (n = 3) independent experiments.See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) FRET signal shows NPC2 binds dehydroergosterol (DHE) with a Kd = 121 ± 75 nM. Data points show mean ± SEM of four (n = 4) independent experiments. (B) NPC2 binds radioactive cholesterol with a Kd = 591 ± 123 nM. Data points show mean ± SD of four (n = 4) independent experiments. (C) NPC2 competition assay. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 by Student’s t test. Data for all radiolabeled binding assays are mean ± SD of five (n = 5) independent experiments. (D) FRET signal shows NTD binds dehydroergosterol (DHE) with a Kd = 483 ± 83 nM. Data points show mean ± SEM of three (n = 3) independent experiments. (E) NTD binds radioactive cholesterol with a Kd = 675 ± 210 nM. Data points show mean ± SD of four (n = 4) independent experiments. (F) NTD competition assay. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; and ∗∗∗∗p ≤ 0.0001 by Student’s t test. Data for all radiolabeled binding assays are mean ± SD of five (n = 5) independent experiments. (G) FRET-based transfer assay of DHE from NTD to NPC2 (full line) and from NPC2 to NTD (dashed line). Data points show mean ± SEM of thr" @default.
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• W2973505555 date "2019-10-01" @default.
• W2973505555 modified "2023-10-10" @default.
• W2973505555 title "Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins" @default.
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• W2973505555 doi "https://doi.org/10.1016/j.cell.2019.08.038" @default.
• W2973505555 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31543266" @default.
• W2973505555 hasPublicationYear "2019" @default.

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