Structure of human Niemann–Pick C1 protein

Abstract

Niemann-Pick C1 protein (NPC1) is a late-endosomal membrane protein involved in trafficking of LDL-derived cholesterol, Niemann-Pick disease type C, and Ebola virus infection. NPC1 contains 13 transmembrane segments (TMs), five of which are thought to represent a "sterol-sensing domain" (SSD). Although present also in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling, the structure and mechanism of action of the SSD are unknown. Here we report a crystal structure of a large fragment of human NPC1 at 3.6 Å resolution, which reveals internal twofold pseudosymmetry along TM 2-13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen. Strikingly, NPC1's SSD forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; computational modeling suggests that this cavity is large enough to accommodate one cholesterol molecule. We propose a model for NPC1 function in cholesterol sensing and transport.endosomal membrane | cholesterol traffic | sterol-sensing domain | crystal structure | allostery C holesterol is a critical component of cellular membranes, and it is either synthesized de novo or supplied from the diet. Although amphiphilic, cholesterol is only poorly soluble in water. Therefore, cholesterol associates with soluble proteins for transport between compartments (1), either as a single molecule or in the form of large lipoprotein particles (2). Cholesterol also functions as a covalently attached ligand in hedgehog-mediated signal transduction (3). Not surprisingly, many proteins involved in cholesterol biosynthesis, transport, or signaling pathways are polytopic, integral membrane proteins (4). Structures of the critical transmembrane region of these polytopic membrane proteins have thus far not been determined, except for an NADPH-dependent reductase in the cholesterol biosynthetic pathway (5), the structure of which yielded insights into the mechanism of intramembrane catalysis.A subgroup of the polytopic integral membrane proteins of cholesterol-related pathways shares a highly conserved region comprised of five transmembrane segments (TMs) that are thought to represent a key regulatory element in response to cholesterol in the bilayer (6). This transmembrane region has been termed the "sterol-sensing domain" (SSD) (7,8). Because crystal structures of these SSDs have not been determined, it has remained unclear precisely how an SSD detects cholesterol in the bilayer and conveys this information to the rest of the protein to influence its activity, stability, or trafficking.Niemann-Pick C1 protein (NPC1) is an SSD-containing, ubiquitous, cholesterol-trafficking protein in the cholesterol uptake pathway (9). Cholesterol is transported throughout the body as cholesterol esters that are packaged into lipoprotein particles including "low-density lipoprotein" (LDL) (2, 10). LDL is endocytosed and transported to late endosomes and lysosomes, where the ∼25-nm particle is subject to lipolysis by lysosomal acid lipase (11,12)...

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

“…Structural insights into NPC1 function have been reported by several groups since 2009: (i) An isolated N-terminal fragment of NPC1 presenting the NTD harbors a cholesterol molecule consistent with previous biochemical observations (19); (ii) a complex of the MLD and Ebola glycoprotein defines the site of Ebola virus attachment (20); (iii) a near-atomic X-ray structure of NPC1 (residues 314-1,278, termed NPC1*) shows that the SSD may indeed accommodate a cholesterol molecule (21); (iv) a cryo-EM…”

“…We expressed and purified the NPC1* (residues 314-1,278) as previously published (21). A previous photo-crosslinking study showed itraconazole binds to NPC1 in vitro (15).…”

Clues to NPC1-mediated cholesterol export from lysosomes

“…Nevertheless, both the N-terminal domain and lumenal domain 2 are likely to interface with NPC2 as part of the cholesterol transfer process (12,13,20). Both the cryo-EM and crystal structures highlight the unusual height of the protein in relation to the lysosome's internal limiting membrane (5,20). This height may serve to provide a cholesterol transfer mechanism that can enable cholesterol to be passed through the glycocalyx that lines the limiting membrane.…”

“…In addition to its important role in cholesterol transport, NPC1 has also been identified as a key component required for the entry of Ebola virus into the cytoplasm (3,4). In PNAS Li et al (5) present a 3D structural model of NPC1, which provides important new clues to the mechanisms by which cholesterol is exported from lysosomes and the mechanism by which Ebola virus docks on the internal surface of the lysosome-limiting membrane as part of the infection process.…”

“…The challenge of obtaining crystals of large, multipass membrane proteins has been aided recently by better protein-expression systems, special detergents, and other novel technologies. Courage is also required, and the fearlessness of Li et al (5) made it possible to obtain a structure model of most of the NPC1 protein with a resolution of 3.6 Å. These authors used a Baculovirusderived system to infect human cells and then isolated NPC1 after membrane solubilization in dodecylmaltoside plus cholesteryl hemisuccinate.…”