title: Characterization of fluorescent sterol binding to purified human NPC1.
abstract: Mutations in the NPC1 gene cause Niemann-Pick type C disease, which appears to result from a defect in intracellular cholesterol trafficking. NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily and contains a sterol-sensing domain, yet its cellular function and the identity of its substrates remain unknown. FLAG-tagged human NPC1 was purified from NPC1-expressing Chinese hamster ovary cells by solubilization in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), followed by affinity chromatography. Purified NPC1 in detergent solution appeared to be oligomeric as determined by gel filtration fast protein liquid chromatography and was photolabeled by an azido-cholesterol derivative. Fluorescent cholesterol analogs, including dehydroergosterol, cholestatrienol, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), displayed enhanced fluorescence upon binding to NPC1 and also resulted in saturable, concentration-dependent quenching of NPC1 intrinsic Trp fluorescence. The apparent binding affinity for these three sterols was in the 0.5-6 microm range. Binding of NBD-cholesterol to NPC1 at low detergent concentration (2 mm CHAPS) was of high apparent affinity (0.5-0.6 microm) and occurred rapidly (<1 min). However, binding of a BODIPY-labeled cholesterol derivative was very slow, requiring approximately 3 h to reach equilibrium. The apparent NBD-cholesterol binding affinity was greatly reduced at higher detergent concentration. The stoichiometry of NBD-cholesterol binding to NPC1 was approximately 1. Various sterols, including native cholesterol and 25-hydroxycholesterol, inhibited NBD-cholesterol binding, suggesting that they compete for binding to the protein. Dynamic quenching studies showed that bound NBD-cholesterol was almost completely shielded from the aqueous medium, suggesting that it is buried in a deep hydrophobic pocket in NPC1. The use of fluorescent cholesterol analogs provides novel information on the molecular properties of the sterol-binding site in the full-length NPC1 protein.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Ronghua
authors.initials: R
authors.lastname: Liu
authors.forename: Peihua
authors.initials: P
authors.lastname: Lu
authors.forename: Joseph W K
authors.initials: JW
authors.lastname: Chu
authors.forename: Frances J
authors.initials: FJ
authors.lastname: Sharom
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002793:Cholic Acids / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006888:Hydroxycholesterols / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D002352:Carrier Proteins; D002793:Cholic Acids; D005456:Fluorescent Dyes; D006888:Hydroxycholesterols; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D011994:Recombinant Proteins; C007997:25-hydroxycholesterol; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate