Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

Abstract

SUMMARY LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3ß-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3ß-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model: after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

“…It has been demonstrated that a water soluble fragment of NPC1 binds cholesterol in an orientation opposite to NPC2 (Infante et al 2008). Based on these results, it has been postulated that, after liberation from LDL, cholesterol is bound by NPC2 which carries it to the lysosomal membrane, where it transfers to the N-terminal domain of the membrane bound NPC1 (Kwon et al 2009). …”

Clues to NPC1-mediated cholesterol export from lysosomes

“…Infante et al discovered that NPC1's N-terminal domain binds cholesterol and this capacity is required for lysosomal cholesterol export (10,11). The N-terminal domain binds cholesterol in opposite orientation from NPC2, suggesting the possibility of a direct hand-off of cholesterol from a soluble NPC2 protein to membrane-associated NPC1 (12). Although this model is very attractive, a direct interaction between the NPC1 N-terminal domain and NPC2 protein has not been detected to date.…”

“…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).…”

“…The ability to generate soluble versions of both the NPC1 N-terminal domain and a lumenal domain 2, facilitated structure determination for these domains (12,18,19). Missing, however, was an understanding of how these domains fit together and how the transmembrane domains are oriented.…”

“…These authors used a Baculovirusderived system to infect human cells and then isolated NPC1 after membrane solubilization in dodecylmaltoside plus cholesteryl hemisuccinate. NPC1's N-terminal domain is attached to the remainder of the protein by a poly-proline-containing flexible arm (12). This flexibility seems to have precluded crystallization of the full-length protein.…”