Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein

Abstract

Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of binding of a lipid to yeast NPC2. We find that NPC2 binds fluorescenceand spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole. 56 oxysterols and, even more weakly, the hydrophobic amine 57 U18666A. 3,9−11 Yeast NPC2 has also been shown to bind 58 cholesterol, ergosterol, DHE, and U18666A, but also 59 edelfosine, a phosphatidylcholine-like lysophospholipid, sug-60 gesting that its binding spectrum is rather broad. 8 While the 61 overall structures of mammalian and yeast NPC2 are similar, 62 the binding site for yeast NPC2 is significantly larger and 63 more open. 8 This difference suggests that yeast NPC2 could 64 eventually bind ligands other than mammalian NPC2.

Photophysical and Structural Characterization of Intrinsically Fluorescent Sterol Aggregates

“…Because titration experiments to assess sterol binding to proteins are typically carried out with DHE concentrations exceeding its CAC in aqueous buffer, sterol self-aggregation must be taken into account in such measurements, typically only providing apparent dissociation constants (discussed in refs and ). To assess sterol–protein interactions, if possible, FRET from aromatic residues of the protein to DHE should be employed as a readout for the binding event. ,,, Alternatively, the increase of DHE fluorescence upon its excitation at ∼330 nm in buffer can be employed as a readout for sterol binding to a given protein. ,,, The fluorescence spectrum of protein-bound DHE when excited at DHE’s maximal absorbance often resembles its spectrum in ethanol (i.e., in the monomeric form), suggesting that many sterol-binding proteins shift the equilibrium between monomer and sterol aggregate in aqueous solution. In fact, Schroeder and co-workers showed that sterol carrier protein 2 not only can bind DHE but also can extract DHE from aggregates in buffer .…”

“…63,67,68,73 Alternatively, the increase of DHE fluorescence upon its excitation at ∼330 nm in buffer can be employed as a readout for sterol binding to a given protein. 15,64,65,74 The fluorescence spectrum of protein-bound DHE when excited at DHE's maximal absorbance often resembles its spectrum in ethanol (i.e., in the monomeric form), suggesting that many sterol-binding proteins shift the equilibrium between monomer and sterol aggregate in aqueous solution. In fact, Schroeder and co-workers showed that sterol carrier protein 2 not only can bind DHE but also can extract DHE from aggregates in buffer.…”

Sterol uptake by the NPC system in eukaryotes: a Saccharomyces cerevisiae perspective

“…NPC2 from S. cerevisiae has demonstrated broad substrate selectivity (binding phosphatidylcholine, phosphatidylinositol and phosphatidylserine). Since the binding of hydrophobic substrates is primarily entropydriven and unspecific, scNPC2 can act as a general lipid solubilizer possibly interacting with several other membrane proteins to shuttle these compounds out of the vacuolar lumen [86,118]. Additionally, atypical NPC2 proteins are shown to shuttle a variety of different sterol types from algal symbionts to their Cnidarian hosts [119], whereas NPC2 from the Japanese carpenter ant has been implicated in binding non-sterol pheromones [83].…”

Mycobacterial membrane protein Large 3‐like‐family proteins in bacteria, protozoa, fungi, plants, and animals: A bioinformatics and structural investigation

“…We thus conclude that the NPC2-like proteins that are found in plant genomes are lipid transporters that are structurally similar to the fungal and animal NPC2s, but there is no evidence that they interact with NCR1 proteins. In fungi, NPC2 can bind to ergosterol as well as PI, PG and phosphatidylcholine 47 and appears to play a role as a multi-lipid transporter: a similar role is likely in plants.…”