Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin

Niemann-Pick Protein C2 ( npc 2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc 2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc 2-membrane interactions has remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc 2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic detail two competitively favorable membrane binding orientations of npc 2 with a low interconversion barrier. The first binding mode ( Prone ) places the cholesterol binding pocket in direct contact with the membrane and is characterized by membrane insertion of a loop (V59-M60-G61-I62-P63-V64-P65). This mode is associated with cholesterol uptake and release. On the other hand, the second mode ( Supine ) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate ( bmp ) is specifically required for strong membrane binding in Prone mode , and that it cannot be substituted by other anionic lipids. Meanwhile, sphingomyelin counteracts bmp by hindering Prone mode without affecting Supine mode . Our results provide concrete evidence that lipids modulate npc 2-mediated cholesterol transport either by favoring or disfavoring Prone mode and that they impose this by modulating the accessibility of bmp for interacting with npc 2. Overall, we provide a mechanism by which npc 2-mediated cholesterol transport is controlled by the membrane composition and how npc 2-lipid interactions can regulate the transport rate.