Niemann–Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding

Abstract

Niemann-Pick type C1 (NPC1) protein is needed for cellular utilization of low-density lipoprotein-derived cholesterol that has been delivered to lysosomes. The protein has 13 transmembrane domains, three large lumenal domains, and a cytoplasmic tail. NPC1's lumenally oriented, N-terminal domain binds cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of the process by which cholesterol is exported from lysosomes into the cytosol. Using surface plasmon resonance and affinity chromatography, we show here that the second lumenal domain of NPC1 binds directly to NPC2 protein. For these experiments, a soluble NPC1 lumenal domain 2 was engineered by replacing adjacent transmembrane domains with antiparallel coiled-coil sequences. Interaction of NPC2 with NPC1 lumenal domain 2 is only detected at acidic pH, conditions that are optimal for cholesterol binding to NPC2 and transfer to NPC1; the pH is also appropriate for the acidic environment where binding would take place. Binding to NPC1 domain 2 requires the presence of cholesterol on NPC2 protein, a finding that supports directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain. Finally, human disease-causing mutations in NPC1 domain 2 decrease NPC2 binding, suggesting that NPC2 binding is necessary for NPC1 function in humans. These data support a model in which NPC1 domain 2 holds NPC2 in position to facilitate directional cholesterol transfer from NPC2 onto NPC1 protein for export from lysosomes.cholesterol trafficking | Niemann-Pick type C disease A major source of cellular cholesterol is endocytosed as lowdensity lipoprotein, which is delivered to late endosomes and lysosomes where cholesterol is released (1). Within late endosomes and lysosomes, Niemann-Pick type C1 (NPC1) and NPC2 proteins are required for the subsequent delivery of cholesterol to other intracellular compartments (2). NPC1 is a large, 1,254 residue, integral membrane protein that is predicted to span the bilayer 13 times (3, 4); it contains a lumenally oriented, N-terminal cholesterol binding site that interacts with the 3β-hydroxyl end of the cholesterol molecule (5-7). NPC2 is a much smaller, soluble lysosomal protein of 132 amino acid residues (8) that binds cholesterol in an opposite orientation via cholesterol's isooctyl side chain (9-11). The importance of NPC1 and NPC2 for cholesterol and glycosphingolipid homeostasis is demonstrated in Niemann-Pick type C disease: patients carrying homozygous mutations in either of these proteins suffer neurodegeneration and die in childhood due to cholesterol and glycosphingolipid accumulation in the brain, liver, and lungs (12, 13).NPC2 is thought to play an important role in extracting cholesterol from intralysosomal membranes that are rich in cholesterol and bis(monoacylglycerol) phosphate. Transfer of that cholesterol to NPC1 is proposed to facilitate passage of this hydrophobic sterol across the glycocalyx that lines the inner lysosomal membrane.Recent studies using purified NPC2 and a soluble con...

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

“…The functions of the NTD and the MLD have been well established by previous studies (18,30,31). Our higher resolution data revealed several hitherto unknown contacts between the CTD and NTD domains of NPC1 and allowed an atomic model for mapping NPC-causing mutations.…”

Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2

“…The NPC2-NPC1 MLD complex structure explains the previously reported cholesterol dependence of this interaction (8).…”

“…These data support a "hydrophobic hand-off" transfer model in which NPC2 binds cholesterol in the lysosome lumen and then binds to NPC1 to deliver cholesterol to NPC1's NTD; transfer of NTD-bound cholesterol to NPC1's transmembrane domain follows, to achieve sterol export from lysosomes (14). Also supporting this transfer model was our finding that NPC2-bearing cholesterol can bind to NPC1's MLD at acidic pH (8). The presence of a conserved, "sterol-sensing" domain within NPC1's membrane spanning portions has implicated these sequences as possible sterol acceptors (15,16).…”

Clues to NPC1-mediated cholesterol export from lysosomes

“…The capped form may represent an inactive state of NPC1 protein because surface residues needed for interaction with NPC2 (and Ebola virus) are shielded by the presence of the N-terminal domain. 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).…”

“…Although this model is very attractive, a direct interaction between the NPC1 N-terminal domain and NPC2 protein has not been detected to date. Using an engineered, soluble construct, Deffieu and Pfeffer (13) showed that NPC1's second lumenal domain binds NPC2 directly, but only at the low pH seen in endosomes and lysosomes and only when it carries cholesterol (13). Thus, the second domain of NPC1 might hold onto NPC2 to facilitate cholesterol transfer from NPC2 to the NPC1 N-terminal domain.…”