Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

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.

[1]  H. Davis,et al.  Ezetimibe, a Potent Cholesterol Absorption Inhibitor, Inhibits the Development of Atherosclerosis in ApoE Knockout Mice , 2001, Arteriosclerosis, thrombosis, and vascular biology.

[2]  Peter L Lee,et al.  Quantitative Measurement of Cholesterol in Cell Populations Using Flow Cytometry and Fluorescent Perfringolysin O. , 2017, Methods in molecular biology.

[3]  S. Pfeffer,et al.  Niemann–Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding , 2011, Proceedings of the National Academy of Sciences.

[4]  Ling Qi,et al.  Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD , 2018, Nature Communications.

[5]  E. Coutavas,et al.  Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex , 2018, Science.

[6]  Torsten Schwede,et al.  SWISS-MODEL: homology modelling of protein structures and complexes , 2018, Nucleic Acids Res..

[7]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[8]  H. Watari,et al.  Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[10]  Gernot Kieseritzky,et al.  Optimizing pKA computation in proteins with pH adapted conformations , 2008, Proteins.

[11]  D. Lairon,et al.  NPC1L1 and SR-BI are Involved in Intestinal Cholesterol Absorption from Small-Size Lipid Donors , 2008, Lipids.

[12]  S. Pfeffer,et al.  NPC intracellular cholesterol transporter 1 (NPC1)-mediated cholesterol export from lysosomes , 2019, The Journal of Biological Chemistry.

[13]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[14]  Nadia Elghobashi-Meinhardt,et al.  Computational Tools Unravel Putative Sterol Binding Sites in the Lysosomal NPC1 Protein , 2019, J. Chem. Inf. Model..

[15]  J. Deisenhofer,et al.  The Structure of the NPC1L1 N-Terminal Domain in a Closed Conformation , 2011, PloS one.

[16]  Luquan Wang,et al.  Materials and Methods Figs. S1 to S4 Tables S1 and S2 References Niemann-pick C1 like 1 Protein Is Critical for Intestinal Cholesterol Absorption , 2022 .

[17]  Dennis C. Ko,et al.  Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Scott,et al.  Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events. , 2001, Molecular biology of the cell.

[19]  Jian Li,et al.  Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells , 2015, Proceedings of the National Academy of Sciences.

[20]  L. Jia,et al.  Niemann-pick C1-like 1 (NPC1L1) protein in intestinal and hepatic cholesterol transport. , 2011, Annual review of physiology.

[21]  Joseph L. Goldstein,et al.  Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol , 2009, Cell.

[22]  Liang Ge,et al.  The N-terminal Domain of NPC1L1 Protein Binds Cholesterol and Plays Essential Roles in Cholesterol Uptake* , 2011, The Journal of Biological Chemistry.

[23]  M. Brown,et al.  A receptor-mediated pathway for cholesterol homeostasis. , 1986, Science.

[24]  B. Cravatt,et al.  Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells , 2013, Nature Methods.

[25]  Ann M Stock,et al.  Structure of a cholesterol-binding protein deficient in Niemann–Pick type C2 disease , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[27]  A. Beaudet,et al.  Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease. , 1975, The Journal of biological chemistry.

[28]  Lisa N Kinch,et al.  Purified NPC1 Protein , 2008, Journal of Biological Chemistry.

[29]  G. Blobel,et al.  Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2 , 2016, Proceedings of the National Academy of Sciences.

[30]  J. Clader,et al.  Discovery of 1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-(4 -hydroxyphenyl)-2-azetidinone (SCH 58235): a designed, potent, orally active inhibitor of cholesterol absorption. , 1998, Journal of medicinal chemistry.

[31]  R. Schmid,et al.  Lipid–Protein Interactions in Niemann–Pick Type C Disease: Insights from Molecular Modeling , 2019, International journal of molecular sciences.

[32]  J. Goldstein,et al.  Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells , 2013, Proceedings of the National Academy of Sciences.

[33]  G. Blobel,et al.  3.3 Å structure of Niemann–Pick C1 protein reveals insights into the function of the C-terminal luminal domain in cholesterol transport , 2017, Proceedings of the National Academy of Sciences.

[34]  J. D. De Brabander,et al.  Structural basis for itraconazole-mediated NPC1 inhibition , 2020, Nature Communications.

[35]  K. Büssow,et al.  Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx , 2012, BMC Biology.

[36]  Amitava Roy,et al.  Detection of long-range concerted motions in protein by a distance covariance. , 2012, Journal of chemical theory and computation.

[37]  Hiroshi Suzuki,et al.  Niemann-Pick C1-Like 1 Overexpression Facilitates Ezetimibe-Sensitive Cholesterol and β-Sitosterol Uptake in CaCo-2 Cells , 2006, Journal of Pharmacology and Experimental Therapeutics.

[38]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[39]  J. Goldstein,et al.  Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1 , 2018, eLife.

[40]  W. Bildl,et al.  Extracellular loop C of NPC1L1 is important for binding to ezetimibe , 2008, Proceedings of the National Academy of Sciences.

[41]  J. Goldstein,et al.  Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection , 2015, eLife.

[42]  Qiang Zhou,et al.  Structural basis for the recognition of Sonic Hedgehog by human Patched1 , 2018, Science.

[43]  Ann M Stock,et al.  Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease* , 2007, Journal of Biological Chemistry.

[44]  P. Pentchev.  Niemann-Pick C research from mouse to gene. , 2004, Biochimica et biophysica acta.

[45]  Amitava Roy,et al.  Analysis of Multidomain Protein Dynamics. , 2016, Journal of chemical theory and computation.

[46]  Andrei L. Lomize,et al.  OPM: Orientations of Proteins in Membranes database , 2006, Bioinform..

[47]  G. Gao,et al.  Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection , 2016, Cell.

[48]  N. Bradbury,et al.  Analysis of CFTR endocytosis by cell surface biotinylation. , 2002, Methods in molecular medicine.

[49]  S. Pfeffer,et al.  Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis , 2016, Molecular biology of the cell.

[50]  Hang Shi,et al.  Structure of human Niemann–Pick C1 protein , 2016, Proceedings of the National Academy of Sciences.

[51]  Daniel Wüstner,et al.  Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins , 2019, Cell.

[52]  Taehoon Kim,et al.  CHARMM‐GUI: A web‐based graphical user interface for CHARMM , 2008, J. Comput. Chem..

[53]  E. Coutavas,et al.  Structures of human Patched and its complex with native palmitoylated sonic hedgehog , 2018, Nature.

[54]  C. Farley,et al.  Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663 , 2000, British journal of pharmacology.