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BackgroundAt the forefront of ecosystems adversely affected by climate change, coral reefs are sensitive to anomalously high temperatures which disassociate (bleaching) photosynthetic symbionts (Symbiodinium) from coral hosts and cause increasingly frequent and severe mass mortality events. Susceptibility to bleaching and mortality is variable among corals, and is determined by unknown proportions of environmental history and the synergy of Symbiodinium- and coral-specific properties. Symbiodinium live within host tissues overlaying the coral skeleton, which increases light availability through multiple light-scattering, forming one of the most efficient biological collectors of solar radiation. Light-transport in the upper ~200 μm layer of corals skeletons (measured as ‘microscopic’ reduced-scattering coefficient, ), has been identified as a determinant of excess light increase during bleaching and is therefore a potential determinant of the differential rate and severity of bleaching response among coral species.ResultsHere we experimentally demonstrate (in ten coral species) that, under thermal stress alone or combined thermal and light stress, low- corals bleach at higher rate and severity than high- corals and the Symbiodinium associated with low- corals experience twice the decrease in photochemical efficiency. We further modelled the light absorbed by Symbiodinium due to skeletal-scattering and show that the estimated skeleton-dependent light absorbed by Symbiodinium (per unit of photosynthetic pigment) and the temporal rate of increase in absorbed light during bleaching are several fold higher in low- corals.ConclusionsWhile symbionts associated with low- corals receive less total light from the skeleton, they experience a higher rate of light increase once bleaching is initiated and absorbing bodies are lost; further precipitating the bleaching response. Because microscopic skeletal light-scattering is a robust predictor of light-dependent bleaching among the corals assessed here, this work establishes as one of the key determinants of differential bleaching response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12898-016-0061-4) contains supplementary material, which is available to authorized users.

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Tissue transglutaminase (TG2) is a multifunctional protein with enzymatic, GTP-ase, and scaffold properties. TG2 interacts with fibronectin (FN) through its N-terminus domain, stabilizing integrin complexes, which regulate cell adhesion to the matrix. Through this mechanism, TG2 participates in key steps involved in metastasis in ovarian and other cancers. High-throughput screening identified several small molecule inhibitors (SMI) for the TG2/FN complex. Rational medicinal chemistry optimization of the hit compound (TG53) led to second-generation analogues (MT1-6). ELISA demonstrated that these analogues blocked TG2/FN interaction, and bio-layer interferometry (BLI) showed that the SMIs bound to TG2. The compounds also potently inhibited cancer cell adhesion to FN and decreased outside-in signaling mediated through the focal adhesion kinase. Blockade of TG2/FN interaction by the small molecules caused membrane ruffling, delaying the formation of stable focal contacts and mature adhesions points and disrupted organization of the actin cytoskeleton. In an in vivo model measuring intraperitoneal dissemination, MT4 and MT6 inhibited the adhesion of ovarian cancer cells to the peritoneum. Pretreatment with MT4 also sensitized ovarian cancer cells to paclitaxel. The data support continued optimization of the new class of SMIs that block the TG2/FN complex at the interface between cancer cells and the tumor niche.

The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)).

Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.

As a novel approach to the structural and functional properties that give rise to extremely stringent sequence specificity in protein-DNA interactions, we have exploited “promiscuous” mutants of EcoRI endonuclease to study the detailed mechanism by which changes in a protein can relax specificity. The A138T promiscuous mutant protein binds more tightly to the cognate GAATTC site than does wild-type EcoRI, yet displays relaxed specificity deriving from tighter binding and faster cleavage at EcoRI* sites (one incorrect base pair). AAATTC EcoRI* sites are cleaved by A138T up to 170-fold faster than by wild type enzyme if the site is abutted by a 5’-purine-pyrimidine (5’-RY) motif. When wild-type protein binds to an EcoRI* site, it forms structurally adapted complexes with thermodynamic parameters of binding that differ markedly from those of specific complexes. By contrast, we show that A138T complexes with 5’-RY-flanked AAATTC sites are virtually indistinguishable from wild-type specific complexes with respect to the heat capacity change upon binding (ΔC°P), the change in excluded macromolecular volume upon association, and contacts to the phosphate backbone. While the preference for the 5’-RY motif implicates contacts to flanking bases as important for relaxed specificity, local effects are not sufficient to explain the large differences in ΔC°P and excluded volume, as these parameters report on global features of the complex. Our findings therefore support the view that specificity does not derive from the additive effects of individual interactions, but rather from a set of cooperative events that are uniquely associated with specific recognition.

We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains. Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall. Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin. In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins. Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains. Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin. The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation.