Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.
WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD 6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer-and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD 6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.
BackgroundInteractions between the orexin peptides and their cognate OX1 and OX2 receptors remain poorly characterized. Site-directed mutagenesis studies on orexin peptides and receptors have indicated amino acids important for ligand binding and receptor activation. However, a better understanding of specific pairwise interactions would benefit small molecule discovery.ResultsWe constructed a set of three-dimensional models of the orexin 1 receptor based on the 3D-structures of the orexin 2 receptor (released while this manuscript was under review), neurotensin receptor 1 and chemokine receptor CXCR4, conducted an exhaustive docking of orexin-A16–33 peptide fragment with ZDOCK and RDOCK, and analyzed a total of 4301 complexes through multidimensional scaling and clustering. The best docking poses reveal two alternative binding modes, where the C-terminus of the peptide lies deep in the binding pocket, on average about 5–6 Å above Tyr6.48 and close to Gln3.32. The binding modes differ in the about 100° rotation of the peptide; the peptide His26 faces either the receptor’s fifth transmembrane helix or the seventh helix. Both binding modes are well in line with previous mutation studies and partake in hydrogen bonding similar to suvorexant.ConclusionsWe present two binding modes for orexin-A into orexin 1 receptor, which help rationalize previous results from site-directed mutagenesis studies. The binding modes should serve small molecule discovery, and offer insights into the mechanism of receptor activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-015-0036-2) contains supplementary material, which is available to authorized users.
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