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      3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport

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          The Niemann–Pick C1 (NPC1) protein is responsible for transporting LDL-derived cholesterol out of late endosomes. Mutations in NPC1 lead to the fatal Niemann–Pick Type C disease. We present here an improved structure of an NPC1 protein at 3.3 Å and decipher details of its C-terminal luminal domain (CTD), which could not be resolved in previous structures. In particular, a loop stabilized by a pair of disulfide bonds in the CTD binds to the N-terminal domain through a loop–loop interaction. We show that this interaction is important for cholesterol transport in cultured cells. Together, our data provide insights related to the molecular mechanism of NPC1 activity and Niemann–Pick Type C disease.


          Niemann–Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann–Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314–1,278), which—in contrast to previous lower resolution structures—features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909–C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD–NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

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          Most cited references 17

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          Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

           Y Ioannou,  D Krizman,  J Gu (1997)
          Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.
            • Record: found
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            Identification of HE1 as the second gene of Niemann-Pick C disease.

            Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.
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              Ebola virus entry requires the host-programmed recognition of an intracellular receptor.

              Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.

                Author and article information

                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                22 August 2017
                7 August 2017
                7 August 2017
                : 114
                : 34
                : 9116-9121
                aLaboratory of Cell Biology, The Rockefeller University , New York, NY 10065;
                bHoward Hughes Medical Institute, The Rockefeller University , New York, NY 10065;
                cDepartment of Molecular Genetics, University of Texas Southwestern Medical Center , Dallas, TX 75390;
                dDepartment of Cell Biology, University of Texas Southwestern Medical Center , Dallas, TX 75390;
                eState Key Laboratory of Membrane Biology, Center for Structural Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China
                Author notes
                2To whom correspondence may be addressed. Email: xli05@ 123456rockefeller.edu or blobel@ 123456rockefeller.edu .

                Contributed by Günter Blobel, July 2, 2017 (sent for review December 19, 2016; reviewed by Daniel S. Ory and Yong Xiong)

                Author contributions: X.L. and G.B. designed research; X.L., F.L., M.N.T., P.S., and J.S. performed research; X.L., F.L., M.N.T., J.W., and G.B. analyzed data; and X.L., F.L., M.N.T., P.S., and G.B. wrote the paper.

                Reviewers: D.S.O., Washington University School of Medicine; and Y.X., Yale University.

                1X.L., F.L., and M.N.T. contributed equally to this work.

                3Present address: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390.

                PMC5576846 PMC5576846 5576846 201711716

                Freely available online through the PNAS open access option.

                Page count
                Pages: 6
                Funded by: Gordon and Betty Moore Foundation (Gordon E. and Betty I. Moore Foundation) 100000936
                Award ID: NA
                Funded by: Howard Hughes Medical Institute (HHMI) 100000011
                Award ID: NA
                Funded by: HHS | NIH | National Institute of General Medical Sciences (NIGMS) 100000057
                Award ID: GM096070
                Funded by: HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI) 100000050
                Award ID: HL20948
                Biological Sciences
                Biophysics and Computational Biology


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