Structure and dynamics of the staphylococcal pyridoxal 5-phosphate synthase complex reveal transient interactions at the enzyme interface.

Infectious diseases are a significant cause of death, and recent studies estimate that common bacterial infectious diseases were responsible for 13.6% of all global deaths in 2019. Among the most significant bacterial pathogens is Staphylococcus aureus, accounting for more than 1.1 million deaths worldwide in 2019. Vitamin biosynthesis has been proposed as a promising target for antibacterial therapy. Here, we investigated the biochemical, structural, and dynamic properties of the enzyme complex responsible for vitamin B6 (pyridoxal 5-phosphate, PLP) biosynthesis in S. aureus, which comprises enzymes SaPdx1 and SaPdx2. The crystal structure of the 24-mer complex of SaPdx1-SaPdx2 enzymes indicated that the S. aureus PLP synthase complex forms a highly dynamic assembly with transient interaction between the enzymes. Solution scattering data indicated that SaPdx2 typically binds to SaPdx1 at a substoichiometric ratio. We propose a structure-based view of the PLP synthesis mechanism initiated with the assembly of SaPLP synthase complex that proceeds in a highly dynamic interaction between Pdx1 and Pdx2. This interface interaction can be further explored as a potentially druggable site for the design of new antibiotics.

Ligand binding free energy evaluation by Monte Carlo Recursion.

The correct evaluation of ligand binding free energies by computational methods is still a very challenging active area of research. The most employed methods for these calculations can be roughly classified into four groups: (i) the fastest and less accurate methods, such as molecular docking, designed to sample a large number of molecules and rapidly rank them according to the potential binding energy; (ii) the second class of methods use a thermodynamic ensemble, typically generated by molecular dynamics, to analyze the endpoints of the thermodynamic cycle for binding and extract differences, in the so-called 'end-point' methods; (iii) the third class of methods is based on the Zwanzig relationship and computes the free energy difference after a chemical change of the system (alchemical methods); and (iv) methods based on biased simulations, such as metadynamics, for example. These methods require increased computational power and as expected, result in increased accuracy for the determination of the strength of binding. Here, we describe an intermediate approach, based on the Monte Carlo Recursion (MCR) method first developed by Harold Scheraga. In this method, the system is sampled at increasing effective temperatures, and the free energy of the system is assessed from a series of terms W(b,T), computed from Monte Carlo (MC) averages at each iteration. We show the application of the MCR for ligand binding with datasets of guest-hosts systems (N = 75) and we observed that a good correlation is obtained between experimental data and the binding energies computed with MCR. We also compared the experimental data with an end-point calculation from equilibrium Monte Carlo calculations that allowed us to conclude that the lower-energy (lower-temperature) terms in the calculation are the most relevant to the estimation of the binding energies, resulting in similar correlations between MCR and MC data and the experimental values. On the other hand, the MCR method provides a reasonable view of the binding energy funnel, with possible connections with the ligand binding kinetics, as well. The codes developed for this analysis are publicly available on GitHub as a part of the LiBELa/MCLiBELa project (https://github.com/alessandronascimento/LiBELa).

Conservation and divergence of the G-interfaces of Drosophila melanogaster septins.

Septins possess a conserved guanine nucleotide-binding (G) domain that participates in the stabilization of organized hetero-oligomeric complexes which assemble into filaments, rings and network-like structures. The fruit fly, Drosophila melanogaster, has five such septin genes encoding Sep1, Sep2, Sep4, Sep5 and Pnut. Here, we report the crystal structure of the heterodimer formed between the G-domains of Sep1 and Sep2, the first from an insect to be described to date. A G-interface stabilizes the dimer (in agreement with the expected arrangement for the Drosophila hexameric particle) and this bears significant resemblance to its human counterparts, even down to the level of individual amino acid interactions. On the other hand, a model for the G-interface formed between the two copies of Pnut which occupy the centre of the hexamer, shows important structural differences, including the loss of a highly favourable bifurcated salt-bridge network. Whereas wild-type Pnut purifies as a monomer, the reintroduction of the salt-bridge network results in stabilizing the dimeric interface in solution as shown by size exclusion chromatography and thermal stability measurements. Adaptive steered molecular dynamics reveals an unzipping mechanism for dimer dissociation which initiates at a point of electrostatic repulsion within the switch II region. Overall, the data contribute to a better understanding of the molecular interactions involved in septin assembly/disassembly.

A scalable screening of E. coli strains for recombinant protein expression.

Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems is usually employed. In this scenario, some of the parameters to be optimized include (i) those related to the protein construct, such as the use of a fusion protein, the choice of an N-terminus fusion/tag or a C-terminus fusion/tag; (ii) those related to the expression stage, such as the concentration and selection of inducer agent and temperature expression and (iii) the choice of the host system, which includes the selection of a prokaryotic or eukaryotic cell and the adoption of a strain. The optimization of some of the parameters related to protein expression, stage (ii), is straightforward. On the other hand, the determination of the most suitable parameters related to protein construction requires a new cycle of gene cloning, while the optimization of the host cell is less straightforward. Here, we evaluated a scalable approach for the screening of host cells for protein expression in a structural biology pipeline. We evaluated four Escherichia coli strains looking for the best yield of soluble heterologous protein expression using the same strategy for protein construction and gene cloning and comparing it to our standard strain, Rosetta 2 (DE3). Using a liquid handling device (robot), E. coli pT-GroE, Lemo21(DE3), Arctic Express (DE3), and Rosetta Gami 2 (DE3) strains were screened for the maximal yield of soluble heterologous protein recovery. For the genes used in this experiment, the Arctic Express (DE3) strain resulted in better yields of soluble heterologous proteins. We propose that screening of host cell/strain is feasible, even for smaller laboratories and the experiment as proposed can easily be scalable to a high-throughput approach.

Modulation of beta-amyloid aggregation using ascorbic acid.

Studies have shown that the level of ascorbic acid (AA) is reduced in the brain of Alzheimer's disease (AD) patients. However, its effect on amyloid-ß 1-42 (Aß42) aggregation has not yet been elucidated. Here we investigated for the first time the effect of AA on Aß42 aggregation using fluorescence assay, circular dichroism, atomic force microscopy, isothermal titration calorimetry, ligand docking, and molecular dynamics. Our results showed that the fibril content decreases in the growth phase when the peptides are co-incubated with AA. AA molecules bind to Aß42 peptides with high binding affinity and a binding site for AA between the ß-strands of Aß42 oligomers prevents the stack of adjacent strands. We demonstrate the inhibitory effect of AA on the aggregation of Aß42 and its molecular interactions, which can contribute to the development of an accessible therapy for AD and also to the design of novel drugs for other amyloidogenic diseases.

The ß-lactam ticarcillin is a Staphylococcus aureus UDP-N-acetylglucosamine 2-epimerase binder.

Infectious diseases account for 25% of the causes of death worldwide and this rate is expected to increase due to antibiotic resistance. Among the bacteria associated with healthcare infections, Staphylococcus aureus is a prevalent pathogen and about 50% of the isolates are found to be methicillin-resistant. Here we describe the identification of ticarcillin as a weak binder of the S. aureus UDP-N-acetylglucosamine 2-epimerase. After a docking screening, ticarcillin was identified as a ligand in using the recently proposed isothermal analysis of differential scanning fluorimetry data. Finally, an equilibrium MD simulation confirmed the docking binding mode as a stable pose, with large contributions to the binding energy coming from interactions between Arg206 and Arg207 and the carboxylate groups in ticarcillin.

Structural Dynamics and Perspectives of Vitamin B6 Biosynthesis Enzymes in Plasmodium: Advances and Open Questions.

Malaria is still today one of the most concerning diseases, with 219 million infections in 2019, most of them in Sub-Saharan Africa and Latin America, causing approx. 409,000 deaths per year. Despite the tremendous advances in malaria treatment and prevention, there is still no vaccine for this disease yet available and the increasing parasite resistance to already existing drugs is becoming an alarming issue globally. In this context, several potential targets for the development of new drug candidates have been proposed and, among those, the de novo biosynthesis pathway for the B6 vitamin was identified to be a promising candidate. The reason behind its significance is the absence of the pathway in humans and its essential presence in the metabolism of major pathogenic organisms. The pathway consists of two enzymes i.e. Pdx1 (PLP synthase domain) and Pdx2 (glutaminase domain), the last constituting a transient and dynamic complex with Pdx1 as the prime player and harboring the catalytic center. In this review, we discuss the structural biology of Pdx1 and Pdx2, together with and the understanding of the PLP biosynthesis provided by the crystallographic data. We also highlight the existing evidence of the effect of PLP synthesis inhibition on parasite proliferation. The existing data provide a flourishing environment for the structure-based design and optimization of new substrate analogs that could serve as inhibitors or even suicide inhibitors.

Tetrazoles as PPARγ ligands: A structural and computational investigation.

Diabetes is an important chronic disease affecting about 10% of the adult population in the US and over 420 million people worldwide, resulting in 1.6 million deaths every year, according to the World Health Organization. The most common type of the disease, type 2 diabetes, can be pharmacologically managed using oral hypoglycemic agents or thiazolidinediones (TZDs), such as pioglitazone, which act by activating the Peroxisome Proliferated-Activated Receptor γ. Despite their beneficial effects in diabetes treatment, TZDs like rosiglitazone and troglitazone were withdrawn due to safety reasons, creating a void in the pharmacological options for the treatment of this important disease. Here, we explored a structure-based approach in the screening for new chemical probes for a deeper investigation of the effects of PPARγ activation. A class of tetrazole compounds was identified and the compounds named T1, T2 and T3 were purchased and evaluated for their ability to interact with the PPARγ ligand binding domain (LBD). The compounds were binders with micromolar range affinity, as determined by their IC50 values. A Monte Carlo simulation of the compound T2 revealed that the tetrazole ring makes favorable interaction with the polar arm of the receptor binding pocket. Finally, the crystal structure of the PPARγ-LBD-T2 complex was solved at 2.3 Å, confirming the binding mode for this compound. The structure also revealed that, when the helix H12 is mispositioned, an alternative binding conformation is observed for the ligand suggesting an H12-dependent binding conformation for the tetrazole compound.

Structural and molecular dynamics investigations of ligand stabilization via secondary binding site interactions in Paenibacillus xylanivorans GH11 xylanase.

Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-ß-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.

Essential Metabolic Routes as a Way to ESKAPE From Antibiotic Resistance.

Antibiotic resistance is a worldwide concern that requires a concerted action from physicians, patients, governmental agencies, and academia to prevent infections and the spread of resistance, track resistant bacteria, improve the use of current antibiotics, and develop new antibiotics. Despite the efforts spent so far, the current antibiotics in the market are restricted to only five general targets/pathways highlighting the need for basic research focusing on the discovery and evaluation of new potential targets. Here we interrogate two biosynthetic pathways as potentially druggable pathways in bacteria. The biosynthesis pathway for thiamine (vitamin B1), absent in humans, but found in many bacteria, including organisms in the group of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp.) and the biosynthesis pathway for pyridoxal 5'-phosphate and its vitamers (vitamin B6), found in S. aureus. Using current genomic data, we discuss the possibilities of inhibition of enzymes in the pathway and review the current state of the art in the scientific literature.

Comparative Analysis of Electrostatic Models for Ligand Docking.

The precise modeling of molecular interactions remains an important goal among molecular modeling techniques. Some of the challenges in the field include the precise definition of a Hamiltonian for biomolecular systems, together with precise parameters derived from Molecular Mechanics Force Fields, for example. The problem is even more challenging when interaction energies from different species are computed, such as the interaction energy involving a ligand and a protein, given that small differences must be computed from large energies. Here we evaluated the effects of the electrostatic model for ligand binding energy evaluation in the context of ligand docking. For this purpose, a classical Coulomb potential with distance-dependent dielectrics was compared with a Poisson-Boltzmann (PB) model for electrostatic potential computation, based on DelPhi calculations. We found that, although the electrostatic energies were highly correlated for the Coulomb and PB models, the ligand pose and the enrichment of actual ligands against decoy compounds, were improved when binding energies were computed using PB as compared to the Coulomb model. We observed that the electrostatic energies computed with the Coulomb model were, on average, ten times larger than the energies computed with the PB model, suggesting a strong overestimation of the polar interactions in the Coulomb model. We also found that a slightly smoothed Lennard-Jones potential combined with the PB model resulted in a good compromise between ligand sampling and energetic scoring.

Energy landscape of the domain movement in Staphylococcus aureus UDP-N-acetylglucosamine 2-epimerase.

Staphylococcus aureus is an important cause of resistant healthcare-associated infections. It has been shown that the wall teichoic acid (WTA) may be an important drug target acting on antibiotic-resistant cells. The UDP-N-acetylglucosamine 2-epimerase, MnaA, is one of the first enzymes on the pathway for the biosynthesis of the WTA. Here, detailed molecular dynamics simulations of S. aureus MnaA were used to characterize the conformational changes that occur in the presence of UDP and UDP-GlcNac and also the energetic landscape associated with these changes. Using different simulation techniques, such as ABMD and GAMD, it was possible to assess the energetic profile for the protein with and without ligands in its active site. We found that there is a dynamic energy landscape that has its minimum changed by the presence of the ligands, with a closed structure of the enzyme being more frequently observed for the bound state while the unbound enzyme favors an opened conformation. Further structural analysis indicated that positively charged amino acids associated with UDP and UDP-GlcNac interactions play a major role in the enzyme opening movement. Finally, the energy landscape profiled in this work provides important conclusions for the design of inhibitor candidates targeting S. aureus MnaA.

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides.

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Šresolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite -1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.

Low-resolution envelope, biophysical analysis and biochemical characterization of a short-chain specific and halotolerant carboxylesterase from Bacillus licheniformis.

Esterases are widely applied in industrial processes due to their versatility, regio- and enantioselectivity, lack of cofactors and stability in organic solvents. Bacillus licheniformis, a microorganism frequently used in industrial and biotechnological applications such as dairy, baking, beverage, pulp and paper, detergent and cosmetics production, organic synthesis and waste management, is a promising source of esterases. Here we describe the biochemical and biophysical characterization of B. licheniformis carboxylesterase BlEst1 and its SAXS-derived molecular envelope. BlEst1 has optimal hydrolytic activity against p­nitrophenyl acetate at pH 7.0 and 40 °C. Furthermore, BlEst1 is stable in different organic solvents such as methanol, isopropanol and butanol. The BlEst1 homology model reveals a typical α/ß hydrolase core with an adjacent auxiliary domain, snuggly fitting the experimental low-resolution SAXS molecular envelope. Moreover, BlEst1 maintained considerable part of its activity in the presence of up to 5 M NaCl and its thermal stability was significantly enhanced by the presence of salt, revealing its halotolerant character. The ability to work under harsh conditions makes BlEst1 an interesting candidate for industrial applications.

GH43 endo-arabinanase from Bacillus licheniformis: Structure, activity and unexpected synergistic effect on cellulose enzymatic hydrolysis.

The hydrolysis of the plant biomass provides many interesting opportunities for the generation of building blocks for the green chemistry industrial applications. An important progress has been made for the hydrolysis of the cellulosic component of the biomass while, for the hemicellulosic components, the advances are less straightforward. Here, we describe the cloning, expression and biochemical and structural characterization of BlAbn1, a GH43 arabinanase from Bacillus licheniformis. This enzyme is selective for linear arabinan and efficiently hydrolyzes this substrate, with a specific activity of 127 U/mg. The enzyme has optimal conditions for activity at pH 8.0 and 45 °C and its activity is only partially dependent of a bound calcium ion since 70% of the maximal activity is preserved even when 1 mM EDTA is added to the reaction medium. BlAbn1 crystal structure revealed a typical GH43 fold and narrow active site, which explains the selectivity for linear substrates. Unexpectedly, the enzyme showed a synergic effect with the commercial cocktail Accellerase 1500 on cellulose hydrolysis. Scanning Electron Microscopy, Solid-State NMR and relaxometry data indicate that the enzyme weakens the interaction between cellulose fibers in filter paper, thus providing an increased access to the cellulases of the cocktail.

Structural and biochemical characterization of a GH3 ß-glucosidase from the probiotic bacteria Bifidobacterium adolescentis.

Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. ß-glucosidases (EC 3.2.1.21) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 ß-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-ß-d-glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-ß-d-xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 Šresolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/ß sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.

Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members.

The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of ß-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.

Structural insights into ß-glucosidase transglycosylation based on biochemical, structural and computational analysis of two GH1 enzymes from Trichoderma harzianum.

ß-glucosidases are glycoside hydrolases able to cleave small and soluble substrates, thus producing monosaccharides. These enzymes are distributed among families GH1, GH2, GH3, GH5, GH9, GH30 and GH116, with GH1 and GH3 being the most relevant families with characterized enzymes to date. A recent transcriptomic analysis of the fungus Trichoderma harzianum, known for its increased ß-glucosidase activity as compared to Trichoderma reesei, revealed two enzymes from family GH1 with high expression levels. Here we report the cloning, recombinant expression, purification and crystallization of these enzymes, ThBgl1 and ThBgl2. A close inspection of the enzymatic activity of these enzymes surprisingly revealed a marked difference between them despite the sequence similarity (53%). ThBgl1 has an increased tendency to catalyze transglycosylation reaction while ThBgl2 acts more as a hydrolyzing enzyme. Detailed comparison of their crystal structures and the analysis of the molecular dynamics simulations reveal the presence of an asparagine residue N186 in ThBgl2, which is replaced by the phenylalanine F180 in ThBgl1. This single amino acid substitution seems to be sufficient to create a polar environment that culminates with an increased availability of water molecules in ThBgl2 as compared to ThBgl1, thus conferring stronger hydrolyzing character to the former enzyme.

Structural and biochemical data of Trichoderma harzianum GH1 ß-glucosidases.

Here the statistics concerning X-ray data processing and structure refinement are given, together with the substrate preference analysis for ThBgl1 and ThBgl2. Finally, the analysis of the influence of temperature and pH on the activities of both enzymes are shown.

An alternative conformation of ERß bound to estradiol reveals H12 in a stable antagonist position.

The natural ligand 17ß-estradiol (E2) is so far believed to induce a unique agonist-bound active conformation in the ligand binding domain (LBD) of the estrogen receptors (ERs). Both subtypes, ERα and ERß, are transcriptionally activated in the presence of E2 with ERß being somewhat less active than ERα under similar conditions. The molecular bases for this intriguing behavior are mainly attributed to subtype differences in the amino-terminal domain of these receptors. However, structural details that confer differences in the molecular response of ER LBDs to E2 still remain elusive. In this study, we present a new crystallographic structure of the ERß LBD bound to E2 in which H12 assumes an alternative conformation that resembles antagonist ERs structures. Structural observations and molecular dynamics simulations jointly provide evidence that alternative ERß H12 position could correspond to a stable conformation of the receptor under physiological pH conditions. Our findings shed light on the unexpected role of LBD in the lower functional response of ERß subtype.