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.

SETD3 was recently identified as the histidine methyltransferase responsible for N 3 -methylation of His73 of β-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected β-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing β-actin peptide was found to be the most potent SETD3 inhibitor (IC 50 = 161 nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for β-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3.

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Much effort has been invested in the search for modulators of membrane transport proteins such as P-glycoprotein (P-gp) to improve drug bioavailability and reverse multidrug resistance in cancer. Nonionic surfactants, a class of pharmaceutical excipients, are known to inhibit such proteins, but knowledge about the exact mechanism of this inhibition is scarce. Here, we perform multiscale molecular dynamics simulations of one of these surfactants, polysorbate 20 (PS20), to reveal the behavior of such compounds on the molecular level and thereby discover the molecular mechanism of the P-gp inhibition. We show that the amphiphilic headgroup of PS20 is too hydrophobic to partition in the water phase, which drives the binding of PS20 to the amphiphilic drug-binding domain of P-gp and thereby causes the inhibition of the protein. Based on our findings, we conclude that PS20 primarily inhibits P-gp through direct binding to the drug-binding domain (DBD) from the extracellular leaflet.

SETD3-catalysed N3-methylation of His73 in β-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies,...