Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO${\sb 2}$ to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of ıt Desulfovibrio vulgaris} formate dehydrogenase AB (ıt Dv}FdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine S${\sp {$δ$}}$ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO${\sb 2}$ reduction, together with an increased sensitivity to oxygen inactivation.

Carbohydrate-binding-modules (CBMs) are discrete auxiliary protein modules with a non-catalytic carbohydrate-binding function and that exhibit a great diversity of binding specificities. CBMcarb-DB is a curated database that classifies the three-dimensional structures of CBM–carbohydrate complexes determined by single-crystal X-ray diffraction methods and solution NMR spectroscopy. We designed the database architecture and the navigation tools to query the database with the Protein Data Bank (PDB), UniProtKB, and GlyTouCan (universal glycan repository) identifiers. Special attention was devoted to describing the bound glycans using simple graphical representation and numerical format for cross-referencing to other glycosciences and functional data databases. CBMcarb-DB provides detailed information on CBMs and their bound oligosaccharides and features their interactions using several open-access applications. We also describe how the curated information provided by CBMcarb-DB can be integrated with AI algorithms of 3D structure prediction, facilitating structure–function studies. Also in this chapter, we discuss the exciting convergence of CBMcarb-DB with the glycan array repository, which serves as a valuable resource for investigating the specific binding interactions between glycans and various biomolecular targets. The interaction of the two fields represents a significant milestone in glycosciences. CBMcarb-DB is freely available at https://cbmdb.glycopedia.eu/ and https://cbmcarb.webhost.fct.unl.pt.

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO2 reduction, towards innovative strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be better understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the oxygen sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. Here we present a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO2). This study reveals that O2 inactivation is promoted by the presence of either substrate and involves forming a different species in the active site, captured in the crystal structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyse the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O2 exposure in the presence of either substrate.

The accumulation of manganese ions is crucial for scavenging reactive oxygen species (ROS) and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains: a DNA binding domain, a dimerization domain, and an ancillary domain, both the metal ion activation mechanism and the DNA recognition mechanism remain elusive. In this study, we present biophysical analyses and the structure of the dimerization and DNA binding domains of DR2539 in its holo form and in complex with the 21 bp pseudo-palindromic repeat of the dr1709 promotor region. These findings shed light into the activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix H3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small angle X-ray scattering experiments and supported by AlphaFold modelling provides a model illustrating the conformational changes induced upon metal binding.Competing Interest StatementThe authors have declared no competing interest.

Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.Arsenic contamination of groundwater is among one of the biggest health threats affecting millions of people in the world. There is an urgent need for efficient arsenic biosensors where the use of arsenic metabolizing enzymes can be explored. In this work, we have solved four crystal structures of arsenite oxidase (Aio) in complex with arsenic and antimony oxyanions and the structures determined correspond to intermediate states of the enzymatic mechanism. These structural data were complemented with density-functional theory calculations providing a unique view of the molybdenum active site at different time points that, together with mutagenesis data, enabled to clarify the enzymatic mechanism and the molecular determinants for the oxidation of As(III) to the less toxic As(V) species.

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.

Human papillomavirus (HPV) is a sexually transmissible virus responsible for 5% of global human cancers and associated with 99% of cervical cancer cases. The oncogenic potential of high-risk HPVs is mainly related to the E6 and E7 oncoproteins, which are responsible, at least in part, for inactivating the p53 and pRb tumor suppressor proteins. Due to the critical role of the E6 protein in malignant tumorigenesis, it is widely recognized as a therapeutic target for anti-HPV drug development. Nevertheless, it is required to obtain large amounts of protein with high purity to perform biointeraction studies with the potential inhibitor drugs. In this work, recombinant dual-tagged E6 protein (His6-MBP-E6) was expressed from Escherichia coli (E. coli) cultures and successfully extracted by sonication/ice cycles. Affinity chromatography using MBPtrap columns allowed 85 ± 5% protein recovery with the elimination of major host heterologous proteins in a single fraction. Subsequently, a polishing step was studied by applying anionic exchange (QSepharose), size exclusion (Superdex), or immobilized-metal affinity chromatography (HisTrap). The combination of affinity chromatography with size exclusion or two affinity chromatography techniques allowed us to obtain 82 ± 2% and 94 ± 3%, of highly pure His6-MBP-E6, respectively. Also, the secondary structure of His6-MBP-E6 is preserved in both purification strategies, as appraised by circular dichroism and western-blot studies. Thermal shift assay confirmed the CD results and suggested potential additives for protein stabilization. Altogether, the reproducible strategies established for the purification of His6-MBP-E6 protein could be successfully applied to later perform biointeraction studies and structural characterization of protein–ligand complexes.

TheBacteroides thetaiotaomicronhas developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RGII depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of themoduleat the C-terminal domain, which we designated BT0996C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Among these effectors, several inclusion membrane proteins (Incs) insert in the vacuolar membrane. Here, we show that human cell lines infected by a C. trachomatis strain deficient for Inc CT288/CTL0540 (renamed IncM) displayed less multinucleation than when infected by IncM-producing strains (wild type or complemented). This indicated that IncM is involved in the ability of Chlamydia to inhibit host cell cytokinesis. The capacity of IncM to induce multinucleation in infected cells was shown to be conserved among its chlamydial homologues and appeared to require its two larger regions predicted to be exposed to the host cell cytosol. C. trachomatis-infected cells also displayed IncM-dependent defects in centrosome positioning, Golgi distribution around the inclusion, and morphology and stability of the inclusion. The altered morphology of inclusions containing IncM-deficient C. trachomatis was further affected by depolymerization of host cell microtubules. This was not observed after depolymerization of microfilaments, and inclusions containing wild-type C. trachomatis did not alter their morphology upon depolymerization of microtubules. Overall, these findings suggest that IncM may exert its effector function by acting directly or indirectly on host cell microtubules.

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.

Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.

Abstract The W/SeCys-FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a dimeric periplasmic enzyme that catalyzes the reversible oxidation of formate and reduction of CO2. It belongs to the group of metal-dependent FDHs, with a tungsten at the active site coordinated by two pyranopterin guanine dinucleotides, a selenocysteine, and one labile sulfur atom. FdhAB has a remarkably high activity and unusual tolerance to oxygen, making it an ideal model system to study biological CO2 reduction.

Abstract Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound VIVO2+ and VIVOL2+, where L=2,2?-bipyridine or 1,10-phenanthroline (phen), and of trypsin with VIVO(picolinato)2 and VVO2(phen)+ moieties. Computational studies complete the refinement and shed light on the relevant role of hydrophobic interactions, hydrogen bonds, and microsolvation in stabilizating the structure. Noteworthy is that the trypsin?VVO2(phen) and trypsin?VIVO(OH)(phen) adducts correspond to similar energies, thus suggesting a possible interconversion under physiological/biological conditions. The obtained data support the relevance of hydrolysis of VIV and VV complexes in the several types of binding established with proteins and the formation of different adducts that might contribute to their pharmacological action, and significantly widen our knowledge of vanadium?protein interactions.

Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe–4S]1+ clusters, and full reduction requires Ti(III)–citrate. The redox potentials of the four [4Fe–4S]1+ centers range between −250 and −530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe–4S]1+ clusters, and full reduction requires Ti(III)–citrate. The redox potentials of the four [4Fe–4S]1+ centers range between −250 and −530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.

Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Silk fibroin is a biobased material with excellent biocompatibility and mechanical properties, but its use in bioelectronics is hampered by the difficult dissolution and low intrinsic conductivity. Some ionic liquids are known to dissolve fibroin but removed after fibroin processing. However, ionic liquids and fibroin can cooperatively give rise to functional materials, and there are untapped opportunities in this combination. The dissolution of fibroin, followed by gelation, in designer ionic liquids from the imidazolium chloride family with varied alkyl chain lengths (2–10 carbons) is shown here. The alkyl chain length of the anion has a large impact on fibroin secondary structure which adopts unconventional arrangements, yielding robust gels with distinct hierarchical organization. Furthermore, and due to their remarkable air-stability and ionic conductivity, fibroin ionogels are exploited as active electrical gas sensors in an electronic nose revealing the unravelled possibilities of fibroin in soft and flexible electronics.

Abstract Relative humidity is simultaneously a sensing target and a contaminant in gas and volatile organic compound (VOC) sensing systems, where strategies to control humidity interference are required. An unmet challenge is the creation of gas-sensitive materials where the response to humidity is controlled by the material itself. Here, humidity effects are controlled through the design of gelatin formulations in ionic liquids without and with liquid crystals as electrical and optical sensors, respectively. In this design, the anions [DCA]− and [Cl]− of room temperature ionic liquids from the 1-butyl-3-methylimidazolium family tailor the response to humidity and, subsequently, sensing of VOCs in dry and humid conditions. Due to the combined effect of the materials formulations and sensing mechanisms, changing the anion from [DCA]− to the much more hygroscopic [Cl]−, leads to stronger electrical responses and much weaker optical responses to humidity. Thus, either humidity sensors or humidity-tolerant VOC sensors that do not require sample preconditioning or signal processing to correct humidity impact are obtained. With the wide spread of 3D- and 4D-printing and intelligent devices, the monitoring and tuning of humidity in sustainable biobased materials offers excellent opportunities in e-nose sensing arrays and wearable devices compatible with operation at room conditions.

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.

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.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors—thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug–drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors—thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug–drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

We investigated the binding of the cephalosporin-class drug cefoperazone (CFP) with bovine serum albumin (BSA) using spectroscopic techniques and in silico methods. The aim of this study was to (i) emphasize the importance of correcting for the inner filter effect in this type of study and (ii) understand the binding mechanism of CFP with BSA by addressing protein conformation and plausible binding sites. Formation of the complex was confirmed by UV–visible spectroscopy. Quenching of BSA fluorescence in the presence of CFP was also observed. Because of the high absorption of CFP in the fluorescence emission range of BSA, the fluorescence emission spectra were corrected for the inner filter effect. Fluorescence emission was studied at excitation wavelengths of 280 and 295 nm. The uncorrected data showed a significant contribution of tyrosine at the excitation wavelength of 280 nm; however, after correction, this contribution became negligible. The static-type mechanism was found to be involved in quenching, with almost 1:1 binding between BSA and CFP. Hydrogen bonding and hydrophobic forces were found to dominate the protein–ligand interactions with a slight decrease in the α-helical contents. Synchronous fluorescence spectral data (at Δλ = 15 and 60 nm) were also corrected for the inner filter effect, with the results being similar to those of excitation at 280 and 295 nm. Molecular docking and molecular dynamics (MD) simulation results suggest that, apart from the two known drug binding sites (drug site I and II), one putative binding site (binding site III) located between BSA domains 1 and 3 was also possible for CFP. MD simulations of the previously reported drug binding sites (drug site I and II) and putative binding site III revealed that binding site III showed excellent binding profiles and could be a target for future research related to BSA-drug binding.

The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conserved putative heme-binding proteins that contains a number of members in animal, plant andbacterial species. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL, have been determined in 2006 and 2011 respectively. In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme bound murine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic in nature, was thought to also involve electrostatic interactions between heme propionate groups and positively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1 variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting in Kd values in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMR relaxation time measurements for human HEBP1 describe a rigid globular protein with no change in motional regime upon heme binding. X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the new heme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1 Å). Results from a HSQC spectrum centred on the histidine side chain Nδ-proton region for HEBP2 confirm that HEBP2 does not bind heme via H42 as no chemical shift differences were observed upon heme addition for backbone NH and Nδ protons. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20 years span a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals and a potential link between heme release under oxidative stress and human HEBP1 is also examined using recent data. However, at the present moment, trying to relate function to the involvement of heme or tetrapyrrole binding, specifically, makes little sense with our current biological knowledge and can only be applied to HEBP1, as HEBP2 does not interact with heme. We suggest that it may not be justified to call this very small family of proteins, heme-binding proteins. The family may be more correctly called “the SOUL family of proteins related to cellular fate” as, even though only HEBP1 binds heme tightly, both proteins may be involved in cell survival and/or proliferation.