Conformational switch of NPC1 epitomizes a lumen-to-membrane alternating access model for nonpolar substrates

Niemann-Pick disease type C (NPC) arises from failure of the NPC1 and/or NPC2 proteins to mobilize lysosomal cholesterol, yet the mechanism by which the complex drives sterol translocation lacks a thorough understanding. Here, we capture an NPC1–NPC2 complex conformation at lysosomal pH using a bis-sterol ligand (JM046), fortifying NPC2 engagement with NPC1 and enabling structural characterization of an alternative transport state. In this complex, the sterol moiety is absent from the NPC1 central tunnel, and the neck site adopts an expanded architecture that resembles a previously observed NPC1 conformation at neutral pH, indicating that the tunnel geometry may not be influenced by the pH alone. Instead, we provide evidence that cholesterol occupancy in the neck site is a key determinant of tunnel contraction. Supporting this notion, NPC1 purified from cholesterol-auxotrophic insect cells exhibits a similarly expanded neck conformation. Integrating these structures and molecular dynamics (MD) simulations exploring the directional cholesterol handoff from NPC2 to the NPC1 tunnel, we propose a substrate-induced gating cycle where luminal and membrane-facing openings of NPC1 switch states in a coordinated manner to transfer cholesterol across the hydrophilic glycocalyx, reminiscent of the alternating access mechanism of membrane transporters. This framework synthesizes NPC1 conformational states into a mechanistic basis that may be used to map pathogenic NPC variants to their relevant transport steps.