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Abstract: Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry.

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Abstract: Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+ via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres.

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Abstract: Inositol pentakisphosphate 2-kinase catalyzes the phosphorylation of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate for de novo synthesis of myo-inositol hexakisphosphate. Disruption of inositol pentakisphosphate 2-kinase profoundly influences cellular processes; from nuclear mRNA export and phosphate homeostasis in yeast and plants, to establishment of left-right asymmetry in zebra fish. We elaborate an active site fluorescent probe that allows high throughput screening of Arabidopsis inositol pentakisphosphate 2-kinase. We show that the probe has a binding constant comparable to the Km values of inositol phosphate substrates of this enzyme, and can be used to prospect for novel substrates and inhibitors of inositol phosphate kinases. We identify several micromolar Ki inhibitors and validate this approach by solving the crystal structure of protein in complex with purpurogallin. We additionally solve structures of protein in complexes with epimeric higher inositol phosphates. This probe may find utility in characterization of a wide family of inositol phosphate kinases.

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Abstract: Ferritins are iron storage proteins that overcome the problems of toxicity and poor bioavailability of iron by catalyzing iron oxidation and mineralization through the activity of a diiron ferroxidase site. Unlike in other ferritins, the oxidized di-Fe3+ site of Escherichia coli bacterioferritin (EcBFR) is stable and therefore does not function as a conduit for the transfer of Fe3+ into the storage cavity, but instead acts as a true catalytic cofactor that cycles its oxidation state while driving Fe2+ oxidation in the cavity. Herein, we demonstrate that EcBFR mineralization depends on three aromatic residues near the diiron site, Tyr25, Tyr58, and Trp133, and that a transient radical is formed on Tyr25. The data indicate that the aromatic residues, together with a previously identified inner surface iron site, promote mineralization by ensuring the simultaneous delivery of two electrons, derived from Fe2+ oxidation in the BFR cavity, to the di-ferric catalytic site for safe reduction of O2.

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Abstract: The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the underpinning mechanisms of these interactions are not understood. Here, we provide structural and functional insights into the canonical mucus-binding protein (MUB), a multi-repeat cell-surface adhesin found in Lactobacillus inhabitants of the GI tract. X-ray crystallography together with small-angle X-ray scattering demonstrated a ‘beads on a string’ arrangement of repeats, generating 174 nm long protein fibrils, as shown by atomic force microscopy. Each repeat consists of tandemly arranged Ig- and mucin-binding protein (MucBP) modules. The binding of full-length MUB was confined to mucus via multiple interactions involving terminal sialylated mucin glycans. While individual MUB domains showed structural similarity to fimbrial proteins from Gram-positive pathogens, the particular organization of MUB provides a structural explanation for the mechanisms in which lactobacilli have adapted to their host niche by maximizing interactions with the mucus receptors, potentiating the retention of bacteria within the mucus layer. Together, this study reveals functional and structural features which may affect tropism of microbes across mucus and along the GI tract, providing unique insights into the mechanisms adopted by commensals and probiotics to adapt to the mucosal environment.