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Title: Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease

Abstract

NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two {beta}-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the {beta}-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrancemore » that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929992
Report Number(s):
BNL-80601-2008-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200822%%1147
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282; Journal Issue: 32
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; AFFINITY; AROMATICS; BUILDUP; CATTLE; CHOLESTEROL; CRYSTAL LATTICES; CRYSTAL STRUCTURE; DISEASES; GLYCOPROTEINS; HYDROCARBONS; LIGANDS; LYSOSOMES; MOLDING; MOLECULES; PROTEINS; RANGE; RESIDUES; SPECIFICITY; STEROLS; SULFATES; TUNNELS; UNITS; national synchrotron light source

Citation Formats

Xu,S., Benoff, B., Liou, H., Lobel, P., and Stock, A.. Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease. United States: N. p., 2007. Web. doi:10.1074/jbc.M703848200.
Xu,S., Benoff, B., Liou, H., Lobel, P., & Stock, A.. Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease. United States. doi:10.1074/jbc.M703848200.
Xu,S., Benoff, B., Liou, H., Lobel, P., and Stock, A.. Mon . "Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease". United States. doi:10.1074/jbc.M703848200.
@article{osti_929992,
title = {Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease},
author = {Xu,S. and Benoff, B. and Liou, H. and Lobel, P. and Stock, A.},
abstractNote = {NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two {beta}-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the {beta}-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.},
doi = {10.1074/jbc.M703848200},
journal = {Journal of Biological Chemistry},
number = 32,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • No abstract prepared.
  • Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures revealmore » the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.« less
  • Although the primary defect is yet unknown, Niemann-Pick disease type C is at present best defined by unique abnormalities of intracellular translocation of exogenous cholesterol. A considerable variation in severity of clinical and biochemical expression has been described: nevertheless, our first genetic complementation study including eleven unrelated patients of varying phenotypes indicated that they all belonged to a single complementation group. We recently mapped the gene responsible for this major group to chromosome 18. In a recent meeting, Fensom and associates reported complementation between the cell line from one patient and those from seven other families. In conjunction with linkagemore » studies, we have now expanded our investigation to the study of 28 unrelated patients. We have found that cell lines from 3 of those patients were able to complement the defect (intralysosomal storage of cholesterol evaluated by filipin staining) in cell lines from the main complementation group (25/28), and did not complement among themselves. Two of the patients had presented with a severe rapidly fatal clinical form while the two affected members in the third family had a slow progressive juvenile phenotype. They all belonged to the classical (severe) biochemical phenotype and had different ethnic origin. Since we learned that one of those cases was the same as that studied by Fensom (personal communication), all Niemann-Pick type C patients studied so far can be divided into two complementation groups. Using microsatellite markers in our multiplex family, we could further show that the gene responsible for the second minor complementation group does not map to the location of the major gene.« less
  • Incubation of fibroblasts derived from patients with type-C Niemann-Pick disease with low density lipoprotein results in excessive intracellular accumulation of unesterified cholesterol. Cytochemical techniques revealed that this abnormal cholesterol accumulation is associated not only with a massive storage of cholesterol in lysosomes but also with a premature cholesterol enrichment of the Golgi complex. Cholesterol appeared also in the Golgi complex of some normal fibroblasts after 24 hr of low density lipoprotein loading. These findings indicate that components of the Golgi complex play a role in the intracellular translocation of exogenously derived cholesterol and that disruptions of the cholesterol transport pathwaymore » at the Golgi may, in part, be responsible for the deficiency in cholesterol utilization in type-C Niemann-Pick fibroblasts.« less
  • Although the A and B subtypes of Niemann-Pick disease (NPD) both result from the deficient activity of acid sphingomyelinase and the lysosomal accumulation of sphingomyelin, they have remarkably distinct phenotypes. Type A disease is a fatal neurodegenerative disorder of infancy, whereas type B disease has no neurologic manifestations and is characterized primarily by reticuloendothelial involvement and survival into adulthood. Both disorders are more frequent among individuals of Ashkenazi Jewish ancestry than in the general population. The recent isolation and characterization of cDNA and genomic sequences encoding ASM has facilitated investigation of the molecular lesions causing the NPD subtypes. Total RNAmore » was reverse-transcribed, and the ASm cDNA from an Ashkenazi Jewish type A patient was specifically amplified by the polymerase chain reaction (PCR). Molecular analysis of the PCR products revealed a G{r arrow} T transversion of nucleotide 1,487, which occurred at a CpG dinucleotide and predicted an Arg{r arrow} Leu substitution and that both parents and several other relatives were heterozygous. These findings identify a frequent missense mutation among NPD patients of Ashkenazi Jewish ancestry that results in neuronopathic type A disease when homoallelic and can result in the nonneuronopathic type B phenotype when heteroallelic. The identification of this ASM mutation in Ashkenazi Jewish patients should facilitate the prevention of NPD in this population by carrier detection with molecular diagnostic techniques.« less