Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins

Abstract

Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the vacuolar/lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system, NCR1/NPC2, we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration which clarifies the role of NPC proteins in this essential eukaryotic pathway and which rationalizes mutations in patients with Niemann-Pick disease Type C.

Inter-domain dynamics drive cholesterol transport by NPC1 and NPC1L1 proteins

“…Taken together, our data support a model in which cholesterol can pass directly through NPC1, either to the sterol-sensing domain or all the way through the membrane bilayer. This model is further supported by recent computations and structural experiments predicting a cholesterol transfer path through the center of NPC1 (Elghobashi-Meinhardt, 2019; see also Wheeler et al, 2019, Winkler et al, 2019Long et al, 2020). Because of the importance of cytoplasmically-oriented residues for NPC1 function, we favor a model in which the sterolsensing domain binding site represents a regulatory site and is not necessarily involved in the transport process per se.…”

“…9B). These data provide new information about the structural basis for Ezetimibe interaction with NPC1L1 protein and support a model whereby Ezetimibe blocks NPC1L1 by locking the various domains together, potentially plugging a potential cholesterol channel through the molecule (Winkler, 2019;Long et al, 2020).…”

“…This finding meshes well with our proposal for how Ezetimibe blocks cholesterol transport by NPC1L1 at a similar location in the corresponding structure. Whether cholesterol exits this channel at the sterol-sensing domain (see also Winkler et al, 2019) Plasmids: cDNAs encoding full length mouse NPC1-eGFP and human NPC1L1-eGFP and ΔN-NPC1L1 mutant (lacks residues 18-260) were described (Johnson and Pfeffer, 2016); P251C, L929C, A521C, K1013C, Δ807-811, 807-811Ala mutants of NPC1 and A179K, L427N, N527D, N527K, K533E, E939K, K1026R, L1029N, L987N and R1002E mutants of NPC1L1…”

“…Moreover, the co-crystal structure of NPC2 bound to NPC1's second lumenal domain (X Li et al, 2016b; see also Winkler et al, 2019) provides a valuable starting point for thinking about how cholesterol is likely to be transferred onto NPC1 protein. Despite these important breakthroughs, we still have much to learn about how NPC1 transfers cholesterol across the lysosome membrane after receiving it from NPC2.…”

Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain

“…In 470 this scenario, confinement of the NTD in the upright position assists cholesterol 471 transport, by a mechanism different from the above-described "handover" 472 mechanism [24]. As the glycocalyx is 5-8 nm thick around the protein, the NTD cannot 473 transfer a cholesterol molecule to either the SSD, or directly to the membrane by just Saccharomyces cerevisiae NPC protein NCR1, the NTD is positioned to deliver a 495 cholesterol molecule to the luminal domains, and that cholesterol reaches the membrane 496 via a tunnel pathway between the MLD and CTD [85]. Whether a similar mechanism 497 applies to the mammalian NPC1 remains to be seen, but a conformationally flexible 498 NTD is necessary for cholesterol to get transported via the tunnel mechanism as well.…”