SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

A quantitative linear model accurately (R^2 = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 °C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 °C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55–56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations

BC2L-C N-Terminal Lectin Domain Complexed with Histo Blood Group Oligosaccharides Provides New Structural Information

International audienceLectins mediate adhesion of pathogens to host tissues, filling in a key role in the first steps of infection. Belonging to the opportunistic pathogen Burkholderia cenocepacia, BC2L-C is a superlectin with dual carbohydrate specificity, believed to mediate cross-linking between bacteria and host cells. Its C-terminal domain binds to bacterial mannosides while its N-terminal domain (BCL2-CN) recognizes fucosylated human epitopes. BC2L-CN presents a tumor necrosis factor alpha (TNF-α) fold previously unseen in lectins with a novel fucose binding mode. We report, here, the production of a novel recombinant form of BC2L-CN (rBC2L-CN2), which allowed better protein stability and unprecedented co-crystallization with oligosaccharides. Isothermal calorimetry measurements showed no detrimental effect on ligand binding and data were obtained on the binding of Globo H hexasaccharide and l-galactose. Crystal structures of rBC2L-CN2 were solved in complex with two blood group antigens: H-type 1 and H-type 3 (Globo H) by X-ray crystallography. They provide new structural information on the binding site, of importance for the structural-based design of glycodrugs as new antimicrobials with antiadhesive properties

Structure and engineering of tandem repeat lectins

International audience(100 − 120 words) Through their ability to bind complex glycoconjugates, lectins have unique specificity and potential for biomedical and biotechnological applications. In particular, lectins with short repeated peptides forming carbohydrate-binding domains are not only of high interest for understanding protein evolution but can also be used as scaffold for engineering novel receptors. Synthetic glycobiology now provides the tools for engineering the specificity of lectins as well as their structure, multivalency and topologies. This review focuses on the structure and diversity of two families of tandem-repeat lectins, i.e. β-trefoils and β-propellers, demonstrated as the most promising scaffold for engineering novel lectins

Etudes structurales et fonctionnelles de lectines et d'adhésines chez Pseudomonas aeruginosa

Pseudomonas aeruginosa est un pathogène opportuniste responsable de nombreuses maladies nosocomiales chez les patients immunodéprimés ainsi que d'infections graves chez les patients atteints de la mucoviscidose (CF). La colonisation des voies respiratoires des patients CF est souvent mortelle car une fois installée, cette bactérie est difficile à éradiquer et provoque le déclin des fonctions respiratoires des patients. L'antibiothérapie devient inefficace face au développement de souches multi-résistantes et sa capacité à former un biofilm. L'étape cruciale initiant l'infection ou la formation du biofilm est l'adhésion durant laquelle des interactions spécifiques lectines/oligosaccharides permettent la fixation de la bactérie à la surface de la cellule hôte. Bloquer l'adhésion serait un moyen de lutter contre l'infection. Dans l'approche d'une thérapie anti-adhésive, plusieurs lectines impliquées dans l'adhésion et l'élaboration du biofilm sont prises comme cibles. Dans un premier temps, des lectines fimbriales putatives identifiées récemment, CupB6 et CupE6, ont fait l'objet d'une étude. Des essais d'expression, de purification et de cristallisation ont été réalisés dans l'objectif de résoudre leur structure cristallographique. Une étude visant à identifier le ligand naturel de CupB6 a également été entreprise. Puis une étude a été menée pour caractériser le potentiel d'inhibition de plusieurs molécules dérivées du galactose sur la lectine soluble, PA-IL. Certaines de ces molécules pourraient être utilisées comme glycomimétiques offrant une alternative aux antibiotiques. Une étude par microcalorimétrie et cristallographie aux rayons X a permis d'étudier la spécificité d'une lectine de légumineuse, PELa.P. aeruginosa is an opportunistic pathogenic bacterium responsible for numerous nosocomial infections in immunosuppressed patients. It is the first mortal pathogen in cystic fibrosis (CF) patients. The invasion of the respiratory tract of CF patients by the bacterium is often lethal because it is hard to eradicate and it rapidly impairs the respiratory functions of the patients. None of the current antibiotherapy procedures are efficient against multiresistant, biofilm forming P. aeruginosa. The first step leading to infection or biofilm formation involves the initial adhesion of bacterial cells to the host pulmonary cells via specific lectin/oligosaccharid interactions. Blocking the adhesion would be a way to fight against the infection. The anti-adhesion therapy targets several bacterial lectins involved in adhesiveness and biofilm formation. In this work, the recently identified putative fimbrial lectins CupB6 and CupE6 have been studied. Expression and purification tests followed by crystallization trials have been performed. In parallel, attempts to identify the natural ligand of CupB6 were also carried out. This work also presents a systematic characterization of the inhibitory effects of various galactose-derived molecules on the PA-IL lectin. Some of these molecules could be used as glycomimetic drugs thus offering an interesting alternative to standard antibiotics. Finally, the combination of microcalorimetry together with X-ray crystallography enabled us to gain insights into the ligand specificity of PELa, a legume lectin.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb

International audienc

The Hidden Conformation of Human Histo-blood Group Antigen is a Determinant for Recognition by Pathogen Lectins

International audienceHisto-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavours revealing the unusual distortion of histo-blood group antigens by bacterial and fungal lectins. The Lewis x trisaccharide adopts a rigid closed conformation in solution, whilst crystallography and molecular dynamics reveal several higher energy open con-formations when bound to the Ralstonia solanacearum lectin, which is in agreement with thermodynamic and kinetic measurements. Extensive molecular dynamics simulations confirm rare transient Le x openings in solution, frequently assisted by distortion of the central N-acetyl-glucosamine ring. Additional directed molecular dynamic trajectories revealed the role of a conserved tryptophan residue in guiding the fucose into the binding site. Our findings show that conformational adaptation of oligosaccharides is of paramount importance in cell recognition and should be considered when designing anti-infective glyco-compounds

Induction of rare conformation of oligosaccharide by binding to calcium-dependent bacterial lectin: X-ray crystallography and modelling study

Pathogenic micro-organisms utilize protein receptors (lectins) in adhesion to host tissues, a process that in some cases relies on the interaction between lectins and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analysed by X-ray crystallography the structures of the LecB lectin from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 4% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we propose a general strategy which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors

Architecture and Evolution of Blade Assembly in β-propeller Lectins

International audienceLectins with a β-propeller fold bind glycans on the cell surface through multivalent binding sites and appropriate directionality. These proteins are formed by repeats of short domains, raising questions about evolutionary duplication. However, these repeats are difficult to detect in translated genomes and seldom correctly annotated in sequence databases. To address these issues, we defined the blade signature of the five types of β-propellers using 3D-structural data. With these templates, we predicted 3887 β-propeller lectins in 1889 species and organised this new information in a searchable online database. The data reveals a widespread distribution of β-propeller lectins across species. Prediction also emphasises multiple architectures and led to uncover a novel β-propeller assembly scenario. This was confirmed by producing and characterizing a predicted protein coded in the genome of Kordia zhangzhouensis. The crystal structure shows a new intermediate in the evolution of β-propeller assembly and demonstrates the power of our tool

Prediction and Validation of a Druggable Site on Virulence Factor of Drug Resistant Burkholderia cenocepacia**

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes infections in patients suffering from chronic granulomatous diseases and cystic fibrosis. It displays significant morbidity and mortality due to extreme resistance to almost all clinically useful antibiotics. The bacterial lectin BC2L-C expressed in B. cenocepacia is an interesting drug target involved in bacterial adhesion and subsequent deadly infection to the host. We solved the first high resolution crystal structure of the apo form of the lectin N-terminal domain (BC2L-C-nt) and compared it with the ones complexed with carbohydrate ligands. Virtual screening of a small fragment library identified potential hits predicted to bind in the vicinity of the fucose binding site. A series of biophysical techniques and X-ray crystallographic screening were employed to validate the interaction of the hits with the protein domain. The X-ray structure of BC2L-C-nt complexed with one of the identified active fragments confirmed the ability of the site computationally identified to host drug-like fragments. The fragment affinity could be determined by titration microcalorimetry. These structure-based strategies further provide an opportunity to elaborate the fragments into high affinity anti-adhesive glycomimetics, as therapeutic agents against B. cenocepacia

Adhesive glycostrategies from opportunistic filamentous fungi

International audienc