Gene transfer agents (GTAs) are phage-derived elements that have evolved repeatedly across diverse prokaryotes, where they drive high-frequency horizontal gene transfer (HGT). Here, we demonstrate that the Rhodobacter capsulatus GTA (RcGTA) tailspike protein, TspA, is a potent biofilm-degrading enzyme. Purified TspA is effective at both preventing initial biofilm formation and clearing established, mature biofilms. Crucially, TspA enhances RcGTA-mediated gene transfer, suggesting that this enzyme facilitates GTA navigation through the extracellular matrix. Unlike the permanently anchored tailspikes of canonical phages, TspA possesses a unique β-sandwich N-terminal domain that enables its dissociation from mature particles and engages in biofilm polysaccharide recognition. Our findings indicate that TspA is an evolutionary adaptation used by GTAs to optimize HGT within complex, densely packed microbial biofilm communities.

The production of short-chain fatty acids (SCFAs) by Firmicutes (Bacillota) within the human gastrointestinal tract is recognized as critical for gut health and the progression of a range of disease states. Firmicutes are the most diverse phylum of human gut bacteria, are highly studied, and are often specialized to degrade just a few polysaccharide substrates. Members of the Firmicutes include key bacteria that produce butyrate, an SCFA that is generally not produced by members of the other major phyla. Recently, it was shown that Eubacterium rectale, a widespread member of the Firmicutes belonging to the Clostridiales cluster XIVa, can grow on the unusual but ubiquitous plant-derived sugar SQ using a sulfoglycolytic sulfofructose transaldolase pathway. Here, we show that in addition to SQ, E. rectale can also grow on the SQ glycoside sulfoquinovosyl glycerol (SQGro). The 3D structure of the E. rectale sulfoquinovosidase (SftG) shares strong structural conservation with other carbohydrate-active enzyme family GH31 SQases. Using sequence-similarity networks, we provide new biological context to a conserved domain of unknown function protein SftX belonging to DUF4867, which is conserved in the sulfoglycolytic sulfofructose transaldolase pathway, and determine its 3D structure. Finally, with the aid of a synthetic mini-human microbiome reconstituted in germfree mice, we show that an SQ dietary supplement can rescue E. rectale from population crashes that occur upon switching from a high-fiber to a low-fiber, high-fat diet. This suggests that SQ or SQGro has the potential as a prebiotic for promoting the maintenance of this important butyrate-producing bacterium within the colonic microbiota.

Microviruses are single-stranded DNA viruses infecting bacteria, characterized by T = 1 shells made of single jelly-roll capsid proteins. To understand how microviruses infect their host cells, we have isolated and studied an unusually large microvirus, Ebor. Ebor belongs to the proposed "Tainavirinae" subfamily of Microviridae and infects the model Alphaproteobacterium Rhodobacter capsulatus. Using cryogenic electron microscopy, we show that the enlarged capsid of Ebor is the result of an extended C-terminus of the major capsid protein. The extra packaging space accommodates genes encoding a lytic enzyme and putative methylase, both absent in microviruses with shorter genomes. The capsid is decorated with protrusions at its 3-fold axes, which we show to recognize lipopolysaccharides on the host surface. Cryogenic electron tomography shows that during infection, Ebor attaches to the host cell via five such protrusions. This attachment brings a single pentameric capsomer into close contact with the cell membrane, creating a special vertex through which the genome is ejected. Both subtomogram averaging and single particle analysis identified two intermediates of capsid opening, showing that the interacting penton opens from its center via the separation of individual capsomer subunits. Structural comparison with the model Bullavirinae phage phiX174 suggests that this genome delivery mechanism may be widely present across Microviridae. IMPORTANCE:Tailless Microviridae bacteriophages are major components of the global virosphere. Notably, microviruses are prominent members of the mammalian gut virome, and certain compositions have been linked to serious health disorders; however, a molecular understanding of how they initiate infection of their host remains poorly characterized. We demonstrate that trimeric protrusions located at the corners of a single microvirus capsomer mediate host cell attachment. This interaction triggers opening of the capsomer, driven by separation of subunits from its center, much like flower petals open during blooming. This extensive opening explains how the genome translocation apparatus, along with the genome itself, is able to exit the capsid. "Penton blooming" likely represents a conserved mechanism shared by diverse viruses possessing similar capsid architectures.

Abstract The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.

The selective alpha,beta-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate-active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective alpha,beta-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering. The W113A cyclohexanone dehydrogenase variant had improved substrate scope, i.e., accepting a bulkier compound, compared to wildtype enzyme. Structural analysis of the variant revealed a broader, more open active site, explaining the new substrate scope.

Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

Molecular replacement is a possible route to obtaining initial phasing for an unknown structure from a known, structurally related molecule. Recent years have seen an explosive growth in the number of protein structures solved using this technique. Automated packages can make the application quite straightforward. Progress has been made in the placing of fragments of complexes, and in the use of imprecise models from NMR or homology modelling. Such models have necessitated the development of new approaches to rebuilding and refinement.

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake - the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with K d values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.

Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.

CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349–712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.

BACKGROUND:Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown.METHODS:Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals.RESULTS:The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the β-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin β-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.

Peptides play an important signalling role in Bacillus subtilis, where their uptake by one of two ABC-type oligopeptide transporters, Opp and App, is required for efficient sporulation. Homologues of these transporters in Clostridium difficile have been characterized, but their role, and hence that of peptides, in regulating sporulation in this organism is less clear. Here, the oligopeptide-binding receptor proteins for these transporters, CdAppA and CdOppA, have been purified and partially characterized, and the crystal structure of CdAppA has been determined in an open unliganded form. Peptide binding to either protein could not be observed in Thermofluor assays with a set of ten peptides of varying lengths and compositions. Re-examination of the protein sequences together with structure comparisons prompts the proposal that CdAppA is not a versatile peptide-binding protein but instead may bind a restricted set of peptides. Meanwhile, CdOppA is likely to be the receptor protein for a nickel-uptake system.