Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4′-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin–dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin–dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

Arsenic is a widely distributed environmental toxin whose presence in drinking water poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase from various bacteria catalyses the oxidation of arsenite to arsenate and is being developed as a biosensor for arsenite. The arsenite oxidase from Rhizobium sp. str. NT-26 (a member of the Alphaproteobacteria) is a heterotetramer consisting of a large catalytic subunit (AioA), which contains a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. Stopped-flow spectroscopy and isothermal titration calorimetry (ITC) have been used to better understand electron transfer through the redox-active centres of the enzyme, which is essential for biosensor development. Results show that oxidation of arsenite at the active site is extremely fast with a rate of >4000s−1 and reduction of the electron acceptor is rate-limiting. An AioB-F108A mutation results in increased activity with the artificial electron acceptor DCPIP and decreased activity with cytochrome c, which in the latter as demonstrated by ITC is not due to an effect on the protein-protein interaction but instead to an effect on electron transfer. These results provide further support that the AioB F108 is important in electron transfer between the Rieske subunit and cytochrome c and its absence in the arsenite oxidases from the Betaproteobacteria may explain the inability of these enzymes to use this electron acceptor.

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Molybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO4 2−, WO4 2− and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of <i>Bacteroidetes</i> in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBP_MLG-A protein encoded by the <i>BACOVA_2743</i> gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBP_MLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBP_MLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBP_MLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows <i>Bacteroidetes</i> to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. <b>IMPORTANCE</b> With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBP_MLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBP_MLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of <i>Bacteroidetes</i>, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90% of adenocarcinomas. Additionally, the binding of the TF-antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction display the potential to prevent cancer metastasis. Herein, a multidisciplinary approach, combining the optimized synthesis of a TF-antigen mimetic with NMR, X-ray crystallography methods and isothermal titration calorimetry assays has been employed to unravel the molecular structural details that govern the Gal-3/TF-mimetic interaction. The TF-mimetic presents a binding affinity for Gal-3 similar to the TF-natural antigen and retains the binding epitope and the bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective a decrease in the enthalpic contribution was observed for the Gal-3/TF-mimetic complex, however this behaviour is compensated by a favourable entropy gain. From a structural perspective, these results establish our TF-mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and likely be used to hamper Gal-3-mediated cancer cells adhesion and metastasis.