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The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco--(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds -(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the -(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing -(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on -(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.

Glucansucrase enzymes synthesize high-molecular-mass extracellular -glucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180-N from Lactobacillusreuteri 180 (lacking the N-terminal domain) revealed an elongated overall structure with two remote domains (IV and V) extending away from the core. By contrast, a new crystal form of the -1,6/-1,3 specific glucansucrase GTF180-N shows an approximate 120(o) rotation of domain V about a hinge located between domains IV and V, giving a much more compact structure than before. Positional variability of domain V in solution is confirmed by small angle X-ray scattering experiments and rigid-body ensemble calculations. In addition, small angle X-ray scattering measurements of full-length GTF180 also provide the first structural data for a full-length glucansucrase, showing that the enzyme has an almost symmetric boomerang-like molecular shape, with a bend likely located between domains IV and V. The similar to 700-residue N-terminal domain, which is not present in the crystal structures, extends away from domain V and the catalytic core of the enzyme. We conclude that, as a result of the hinge region, in solution, GTF180-N (and likely also the full-length GTF180) shows conformational flexibility; this may be a general feature of GH70 glucansucrases.Database center dot Structural data for GTF180-N II have been deposited in the Protein Data Bank under accession code .

The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched alpha-glucans with both alpha-1,6-and alpha-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-Delta N, a 118 kDa fragment of GTFA comprising residues 745-1763 and including the catalytic domain, was determined at 3.6 angstrom resolution by molecular replacement. The crystals have large solvent channels and an unusually high solvent content of 85%. GTFA-Delta N has the same domain arrangement and domain topologies as observed in previously determined GH70 glucansucrase structures. The architecture of the GTFA-Delta N active site and binding pocket confirms that glucansucrases have a conserved substrate specificity for sucrose. However, this first crystal structure of an alpha-1,6/alpha-1,4-specific glucansucrase shows that residues from conserved sequence motif IV (1128-1136 in GTFA-Delta N) contribute to the acceptor-binding subsites and that they display differences compared with other structurally characterized glucansucrases. In particular, the structure clarifies the importance of residues following the transition-state stabilizer for product specificity, and especially residue Asn1134, which is in a position to interact with sugar units in acceptor subsite +2.

Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like ( β / α ) 8 -barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a “U”-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product.

Class II phosphoinositide 3-kinases (PI3K-C2) are large multidomain enzymes that control cellular functions ranging from membrane dynamics to cell signaling via synthesis of 3'-phosphorylated phosphoinositides. Activity of the alpha isoform (PI3K-C2α) is associated with endocytosis, angiogenesis, and glucose metabolism. How PI3K-C2α activity is controlled at sites of endocytosis remains largely enigmatic. Here we show that the lipid-binding PX-C2 module unique to class II PI3Ks autoinhibits kinase activity in solution but is essential for full enzymatic activity at PtdIns(4,5)P2-rich membranes. Using HDX-MS, we show that the PX-C2 module folds back onto the kinase domain, inhibiting its basal activity. Destabilization of this intramolecular contact increases PI3K-C2α activity in vitro and in cells, leading to accumulation of its lipid product, increased recruitment of the endocytic effector SNX9, and facilitated endocytosis. Our studies uncover a regulatory mechanism in which coincident binding of phosphoinositide substrate and cofactor selectively activate PI3K-C2α at sites of endocytosis.

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass alpha-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742-1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2(1)2(1)2(1)). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 angstrom resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.

The smallest extrinsic polypeptide of the water-oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn2+ and its structure was determined by X-ray diffraction at 1.95-Å resolution using the multi-wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four-helix bundle, whereas the amino-terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl− and Ca2+ and make them available to PSII.

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway.