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-α-(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 α-(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 α-(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 α-(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 α-(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.
Proline‐specific endoproteases have been successfully used in, for example, the in‐situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the ...generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline‐specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline‐specific endoprotease activity. Family S28 proteases have a conventional Ser‐Asp‐His catalytic triad, but their oxyanion‐stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics‐assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28‐proline‐specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino‐ and carboxyl‐terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity.
The ATP-binding cassette (ABC) transporter GlnPQ is an essential uptake system for amino acids in gram-positive pathogens and related nonpathogenic bacteria. The transporter has tandem ...substrate-binding domains (SBDs) fused to each transmembrane domain, giving rise to four SBDs per functional transporter complex. We have determined the crystal structures and ligand-binding properties of the SBDs of GlnPQ from Enterococcus faecalis, Streptococcus pneumoniae, and Lactococcus lactis. The tandem SBDs differ in substrate specificity and affinity, allowing cells to efficiently accumulate different amino acids via a single ABC transporter. The combined structural, functional, and thermodynamic analysis revealed the roles of individual residues in determining the substrate affinity. We succeeded in converting a low-affinity SBD into a high-affinity receptor and vice versa. Our data indicate that a small number of residues that reside in the binding pocket constitute the major affinity determinants of the SBDs.
•Crystal structures of liganded and unliganded receptor domains•Molecular basis of high-affinity amino acid binding•Tandem ligand-binding domains operate independent from each other•Dual specificity and dual affinity transport
The ATP-binding cassette transporter GlnPQ is an essential uptake system for amino acids in Gram-positive bacteria. Fulyani et al. show that a small number of residues that reside in the binding pocket of the receptor domains of GlnPQ constitute the major affinity determinants for transport.
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.
Display omitted
•C-terminal lipid-binding domains of PI3K-C2α (PX and C2 domains) inhibit activity•HDX-MS identifies inhibitory contact site between PX-C2 module and kinase domain•Single-site mutation at the inhibitory interface hyperactivates PI3K-C2α•Hyperactive PI3K-C2α enhances PtdIns(3,4)P2 cellular formation and endocytosis
Using recombinantly expressed protein, Wang et al. discovered that the lipid kinase activity of PI3K-C2α is inhibited by its C-terminal lipid-binding domains. Disruption of the inhibitory interface by mutation or lipid engagement of the C terminus with PtdIns(4,5)P2 stimulates kinase activity, while cellular hyperactivation of PI3K-C2α enhances PtdIns(3,4)P2 production and endocytosis.
Glucansucrase enzymes synthesize high‐molecular‐mass extracellular α‐glucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180‐ΔN ...from Lactobacillus reuteri 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 α‐1,6/α‐1,3 specific glucansucrase GTF180‐ΔN shows an approximate 120o 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 ~ 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‐ΔN (and likely also the full‐length GTF180) shows conformational flexibility; this may be a general feature of GH70 glucansucrases.
Database
• Structural data for GTF180‐ΔN II have been deposited in the Protein Data Bank under accession code 4AYG.
The structure of N‐terminally truncated glucansucrase GTF180‐ΔN from Lactobacillus reuteri 180 in a second crystal form reveals that domains V can adopt completely different positions. SAXS experiments with GTF180‐ΔN and the full‐length GTF180 confirm this positional variability, and show that conformational flexibility, which may be a general feature of glucansucrases, occurs in solution.
Summary
Glycan utilization plays a key role in modulating the composition of the gut microbiota, but molecular insight into oligosaccharide uptake by this microbial community is lacking. ...Arabinoxylo‐oligosaccharides (AXOS) are abundant in the diet, and are selectively fermented by probiotic bifidobacteria in the colon. Here we show how selectivity for AXOS uptake is established by the probiotic strain Bifidobacterium animalis subsp. lactis Bl‐04. The binding protein BlAXBP, which is associated with an ATP‐binding cassette (ABC) transporter that mediates the uptake of AXOS, displays an exceptionally broad specificity for arabinosyl‐decorated and undecorated xylo‐oligosaccharides, with preference for tri‐ and tetra‐saccharides. Crystal structures of BlAXBP in complex with four different ligands revealed the basis for this versatility. Uniquely, the protein was able to recognize oligosaccharides in two opposite orientations, which facilitates the optimization of interactions with the various ligands. Broad substrate specificity was further enhanced by a spacious binding pocket accommodating decorations at different mainchain positions and conformational flexibility of a lid‐like loop. Phylogenetic and genetic analyses show that BlAXBP is highly conserved within Bifidobacterium, but is lacking in other gut microbiota members. These data indicate niche adaptation within Bifidobacterium and highlight the metabolic syntrophy (cross‐feeding) among the gut microbiota.
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.
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.
•The endocytic membrane-deforming BAR domain protein SNX9 is autoinhibited•Release of SNX9 from autoinhibition requires lipid and endocytic adaptor binding•Coincident detection of lipid and adaptor enables membrane remodeling by SNX9•Restriction of membrane bending by BAR proteins drives progression of endocytosis
Dynamic membrane remodeling is essential for many cell physiological processes, including endocytosis. Lo et al. show that membrane constriction by the endocytic BAR domain protein SNX9 is controlled by coincident detection of the lipid PI(3,4)P2 and the clathrin adaptor AP2.
Energy coupling factor (ECF) transporters are used for the uptake of vitamins in Prokarya. They consist of an integral membrane protein that confers substrate specificity (the S-component) and an ...energizing module that is related to ATP-binding cassette (ABC) transporters. S-components for different substrates often do not share detectable sequence similarity but interact with the same energizing module. Here we present the crystal structure of the thiamine-specific S-component ThiT from Lactococcus lactis at 2.0 Å. Extensive protein-substrate interactions explain its high binding affinity for thiamine (K(d) ~10(-10) M). ThiT has a fold similar to that of the riboflavin-specific S-component RibU, with which it shares only 14% sequence identity. Two alanines in a conserved motif (AxxxA) located on the membrane-embedded surface of the S-components mediate the interaction with the energizing module. Based on these findings, we propose a general transport mechanism for ECF transporters.
Deinococcaceae are famous for their extreme radioresistance. Transcriptome analysis in
Deinococcus radiodurans revealed a group of genes up-regulated in response to desiccation and ionizing ...radiation. IrrE, a novel protein initially found in
D. radiodurans, was shown to be a positive regulator of some of these genes.
Deinococcus deserti irrE is able to restore radioresistance in a
D. radiodurans Δ
irrE mutant. The
D. deserti IrrE crystal structure reveals a unique combination of three domains: one zinc peptidase-like domain, one helix-turn-helix motif and one GAF-like domain. Mutant analysis indicates that the first and third domains are critical regions for radiotolerance. In particular, mutants affected in the putative zinc-binding site are as sensitive to gamma and UV irradiation as the Δ
irrE bacteria, and radioresistance is strongly decreased with the H217L mutation present in the C-terminal domain. In addition, modeling of IrrE–DNA interaction suggests that the observed IrrE structure may not bind double-stranded DNA through its central helix-turn-helix motif and that IrrE is not a classic transcriptional factor that activates gene expression by its direct binding to DNA. We propose that the putative protease activity of IrrE could be a key element of transcription enhancement and that a more classic transcription factor, possibly an IrrE substrate, would link IrrE to transcription of genes specifically involved in radioresistance.
PDF
