Search

Many biological molecules are assembled into supramolecular complexes that are necessary to perform functions in the cell. Better understanding and characterization of these molecular assemblies are thus essential to further elucidate molecular mechanisms and key protein-protein interactions that could be targeted to modulate the protein binding affinity or develop new binders. Experimental access to structural information on these supramolecular assemblies is often hampered by the size of these systems that make their recombinant production and characterization rather difficult. Computational methods combining both structural data, molecular modeling techniques, and sequence coevolution information can thus offer a good alternative to gain access to the structural organization of protein complexes and assemblies. Herein, we present some computational methods to predict structural models of the protein partners, to search for interacting regions using coevolution information, and to build molecular assemblies. The approach is exemplified using a case study to model the succinate-quinone oxidoreductase heterocomplex.

International audience; N-acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant-specific Lysin Motif Receptor-Like Kinases (LysM-RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo-chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM-RLK LYR3 (MtLYR3) as a specific LCO-binding protein. We also showed that the absence of LCO binding to LYR3 of the non-mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site-directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3-LysM3 by those of MtLYR3-LysM3 allowed the recovery of high-affinity LCO binding in experimental radioligand-binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.

International audience; Many biological molecules are assembled into supramolecular complexes that are necessary to perform functions in the cell. Better understanding and characterization of these molecular assemblies are thus essential to further elucidate molecular mechanisms and key protein-protein interactions that could be targeted to modulate the protein binding affinity or develop new binders. Experimental access to structural information on these supramolecular assemblies is often hampered by the size of these systems that make their recombinant production and characterization rather difficult. Computational methods combining both structural data, molecular modeling techniques, and sequence coevolution information can thus offer a good alternative to gain access to the structural organization of protein complexes and assemblies. Herein, we present some computational methods to predict structural models of the protein partners, to search for interacting regions using coevolution information, and to build molecular assemblies. The approach is exemplified using a case study to model the succinate-quinone oxidoreductase heterocomplex.

International audience; The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions. In this work, a Carbohydrate-Binding Module (CBM) was used as a central core to provide a versatile strategy to bring a large diversity of functions to cellulose surfaces. CBM3a from Clostridium thermocellum, which has a high affinity for crystalline cellulose, was flanked through linkers with a streptavidin domain and an azide group introduced through a non-canonical amino acid. Each of these two extra domains was effectively produced and functionalized with a variety of biological and chemical molecules. Structural properties of the resulting tripartite chimeric protein were investigated using molecular modelling approaches, and its potential for the multi-functionalization of cellulose was confirmed experimentally. As a proof of concept, we show that cellulose can be labelled with a fluorescent version of the tripartite protein grafted to magnetic beads and captured using a magnet.

International audience; Multi-component adsorption of proteins still requires a better understanding of local phenomena to improve the development of predictive models. In this work, all-atom Molecular Dynamics (MD) simulations were used to investigate the influence of protein charge distribution on the adsorption capacity. The simultaneous adsorption of alpha-chymotrypsin and lysozyme on a cation exchanger, SP Sepharose FF, was studied through MD simulations and compared to macroscopic isotherm experiments. It appears that the charge distribution is a relevant information to better understand specific phenomena, such as a multilayer adsorption caused by the particular electrostatic profile of alpha-chymotrypsin. Therefore, MD simulations seem to be an interesting way to visualize and highlight these behaviors.

International audience; The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and β-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-β-d-galactofuranosides were increased up to 4.8-fold compared to the wild-type enzyme. In depth characterization revealed that the mutants exhibit increased transfer rates and thus a hydrolysis/self-condensation ratio in favor of synthesis. The consequence of the substitution N216W is the creation of an additional binding subsite that provides the basis for an alternative acceptor substrate binding mode. As a result, mutants bearing N216W synthesize not only (1,2)-linked furanobiosides, but also (1,3)- and even (1,5)-linked furanobiosides. Since the self-condensation is under kinetic control, the yield of homo-disaccharides was maximized using higher substrate concentrations. In this way, the mutant R69H-N216W produced oligo-β-d-galactofuranosides in > 70% yield. Overall, this study further demonstrates the potential usefulness of TxAbf mutants for glycosynthesis and shows how these might be used to synthesize biologically-relevant glycoconjugates.

International audience; Plant a-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by a-galactosidases produced by the human gut microbiome. In this environment, the Ruminococcus gnavus E1 symbiont–well-known for various benefit–is able to produce an original RgAgaSK bifunctional enzyme. This enzyme contains an hydrolytic a-galactosidase domain linked to anATP dependent extra-domain, specifically involved in the a-galactoside hydrolysis and the phosphorylation of the glucose, respectively. However, the multi-modular relationships between both catalytic domains remained hitherto unexplored and has been, consequently, herein investigated. Biochemical characterization of heterologously expressed enzymes either in full-form or in separated domains revealed similar kinetic parameters. These results were supported by molecular modeling studies performed on the whole enzyme in complex with different RFOs. Further enzymatic analysis associated with kinetic degradation of various substrates followed by high pressure anionic exchange chromatography revealed that catalytic efficiency decreased as the number of D-galactosyl moieties branched onto the oligosaccharide increased, suggesting a preference of RgAgaSK for RFO’s short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the RgAgaSK encodinggene whatever the health status of the individuals. Finally, phylogeny and synteny studies suggested a limited spread by horizontal transfer of the clusters’ containing RgAgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.

Funder: Royal Society Newton fellowship, Funder: CAPES Scholarship - Brazil, Background: Despite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota. Results: Here, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for β-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired. Conclusion: By compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships. BEyZgKg3YsWtKJ_ei8gXkiVideo Abstract

The enzymatic transamination of ketones into (

International audience; The enzymatic transamination of ketones into (R)-amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of (R)-selective amine transaminases [(R)-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous a-amino acid transaminases (alpha-AATs). Here, we show the successful introduction of an (R)-transaminase activity in an alpha-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the D-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg(-1) in the conversion of (R)-phenylethylamine and pyruvate to acetophenone and b-alanine. This value is similar to those of natural (R)-ATAs, which typically are in the range of 250 milliunits mg(-1). These results demonstrate that (R)-ATAs can evolve from alpha-AAT as shown here for the DATA scaffold.

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for thein vitrosynthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of theShigellalipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

Proteins of the independent mevalonate pathway for isoprenoid biosynthesis are important targets for the development of new antibacterial compounds as this pathway is present in most pathogenic organisms such as Mycobacterium tuberculosis, DPlasmodium falciparum and Escherichia coli, but is not present in mammalian species, including humans. Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an important target in this pathway and the most effective DXR inhibitor to date is fosmidomycin, which is used to treat malaria and, more recently, tuberculosis. Recently, Armstrong C. M. et al. showed that a mutant of DXR, S222T, induces a loss of the fosmidomycin inhibition efficiency, even though the bacteria culture is still viable and able to produce isoprenoids. As this represents a potential fosmidomycin-resistant mutation, it is important to understand the mechanism of this apparent mutation-induced resistance to fosmidomycin. Here, we used molecular dynamics simulations and Molecular Mechanics/Poisson Boltzmann Surface Area analysis to understand the structural and energetic basis of the resistance. Our results suggest that the point mutation results in changes to the structural dynamics of an active site loop that probably protects the active site and facilitates enzymatic reaction. From the simulation analysis, we also showed that the mutation results in changes in the interaction energy profiles in a way that can explain the observed activity of the mutant protein toward the natural inhibitor deoxy-D-xylulose 5-phosphate. These results should be taken into consideration in future efforts to develop new therapeutic antibiotic compounds that target DXR.

International audience; The interest for a better understanding of ion-exchange mechanisms at the atomic level has strongly increased over the past decades. Indeed, molecular-level information about physico-chemical mechanisms could help optimizing chromatographic processes for protein purification, which are sub-optimized due to the lack of predictive models. A promising approach is based on the use of Molecular Dynamics (MD) simulations to study local phenomena inside adsorbents which can then be challenged against experimental results. In this work, macroscopic experimental data, consisting in the ion-exchange uptake of alpha-chymotrypsin onto SP Sepharose FF, have been compared to the adsorption behavior predicted by MD simulations. The chromatographic surface, represented as a uniform distribution of ligands with a counterion layer, in the presence of the protein was modeled using all-atom representation. The SMA formalism was used to describe single adsorption isotherms and to relate macroscopic observations with molecular simulations. Two SMA parameters based on physical principles, the characteristic charge n and the steric factor sigma, have been estimated by both experiments and MD simulations. At pH 5 and NaCl concentration of 100 mM, our study shows a fairly good agreement between both results, especially for the characteristic charge. It is shown that the steric factor calculation is strongly dependent on the ligand density on the adsorbent surface, whose value must be carefully determined in order to obtain reliable predictions. In addition, four binding patches were identified as being involved in the adsorption, which have been confirmed through binding free energy calculations.

Among alpha-transglucosylases from Glycoside-Hydrolase family 70, the Delta N-123-GB-CD2 enzyme derived from the bifunctional DSR-E from L. citreum NRRL B-1299 is particularly interesting as it was the first described engineered Branching Sucrase, not able to elongate glucan polymers from sucrose substrate. The previously reported overall structural organization of this multi-domain enzyme is an intricate U-shape fold conserved among GH70 enzymes which showed a certain conformational variability of the so-called domain V, assumed to play a role in the control of product structures, in available X-ray structures. Understanding the role of functional dynamics on enzyme reaction and substrate recognition is of utmost interest although it remains a challenge for biophysical methods. By combining long molecular dynamics simulation (1 mu s) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of Delta N-123-GB-CD2 alone and in interaction with sucrose substrate. Overall, our results provide the detailed picture at atomic level of the hierarchy of motions occurring along different timescales and how they are correlated, in agreement with experimental structural data. In particular, detailed analysis of the different structural domains revealed cooperative dynamic behaviors such as twisting, bending and wobbling through anti- and correlated motions, and also two structural hinge regions, of which one was unreported. Several highly flexible loops surrounding the catalytic pocket were also highlighted, suggesting a potential role in the acceptor promiscuity of Delta N-123-GBD-CD2. Normal modes and essential dynamics underlined an interesting two-fold dynamic of the catalytic domain A, pivoting about an axis splitting the catalytic gorge in two parts. The comparison of the conformational free energy landscapes using principal component analysis of the enzyme in absence or in presence of sucrose, also revealed a more harmonic basin when sucrose is bound with a shift population of the bending mode, consistent with the substrate binding event.

Given the tight relation between protein structure and function, we present a set of methods to analyze protein topology, implemented in the VLDP program, relying on Laguerre space partitions built from series of molecular dynamics snapshots. The Laguerre partition specifies inter-atomic contacts, formalized in graphs. The deduced properties are the existence and count of water aggregates, possible passage ways and constrictions, the structure, connectivity, stability and depth of the water network. As a test-case, the membrane protein FepA is investigated in its full environment, yielding a more precise description of the protein surface. Inside FepA, the solvent splits into isolated clusters and an intricate network connecting both sides of the lipid bilayer. The network is dynamic, connections set on and off, occasionally substantially relocating traversing paths. Subtle differences are detected between two forms of FepA, ligand-free and complexed with its natural iron carrier, the enterobactin. The complexed form has more constricted and more centered openings in the upper part whereas, in the lower part, constriction is released: two main channels between the plug and barrel lead directly to the periplasm. Reliability, precision and the variety of topological features are the main interest of the method.

Hydroxamate analogs of fosfoxacin, the phosphate homolog of fosmidomycin, have been synthesized and their activity tested on Escherichia coli and Mycobacterium smegmatis DXRs. Except for compound 4b, the IC50 values of phosphate derivatives are approximately 10-fold higher than those of the corresponding phosphonates. Although their inhibitory activity on Escherichia coli DXR is less efficient than their phosphonate analogs, we report the ability of phosphate compounds to inhibit the growth of Escherichia coli. This work points out that the uptake of fosfoxacin and its analogs is taking place via the GlpT and UhpT transporters. As expected, these compounds are inefficient to inhibit the growth of M. smegmatis growth inhibition probably due to a lack of uptake.

International audience; Transmembrane proteins (TMPs) are major drug targets, but the knowledge of their precise topology structure remains highly limited compared with globular proteins. In spite of the difficulties in obtaining their structures, an important effort has been made these last years to increase their number from an experimental and computational point of view. In view of this emerging challenge, the development of computational methods to extract knowledge from these data is crucial for the better understanding of their functions and in improving the quality of structural models. Here, we revisit an efficient unsupervised learning procedure, called Hybrid Protein Model (HPM), which is applied to the analysis of transmembrane proteins belonging to the all-alpha structural class. HPM method is an original classification procedure that efficiently combines sequence and structure learning. The procedure was initially applied to the analysis of globular proteins. In the present case, HPM classifies a set of overlapping protein fragments, extracted from a non-redundant databank of TMP 3D structure. After fine-tuning of the learning parameters, the optimal classification results in 65 clusters. They represent at best similar relationships between sequence and local structure properties of TMPs. Interestingly, HPM distinguishes among the resulting clusters two helical regions with distinct hydrophobic patterns. This underlines the complexity of the topology of these proteins. The HPM classification enlightens unusual relationship between amino acids in TMP fragments, which can be useful to elaborate new amino acids substitution matrices. Finally, two challenging applications are described: the first one aims at annotating protein functions (channel or not), the second one intends to assess the quality of the structures (X-ray or models) via a new scoring function deduced from the HPM classification.