Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

<p>Abstract</p> <p>Background</p> <p>Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins.</p> <p>Results</p> <p>The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry.</p> <p>Conclusions</p> <p>Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, <it>Phytophthora</it>. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.</p

Molecular basis for bacterial peptidoglycan recognition by LysM domains.

Carbohydrate recognition is essential for growth, cell adhesion and signalling in all living organisms. A highly conserved carbohydrate binding module, LysM, is found in proteins from viruses, bacteria, fungi, plants and mammals. LysM modules recognize polysaccharides containing N-acetylglucosamine (GlcNAc) residues including peptidoglycan, an essential component of the bacterial cell wall. However, the molecular mechanism underpinning LysM-peptidoglycan interactions remains unclear. Here we describe the molecular basis for peptidoglycan recognition by a multimodular LysM domain from AtlA, an autolysin involved in cell division in the opportunistic bacterial pathogen Enterococcus faecalis. We explore the contribution of individual modules to the binding, identify the peptidoglycan motif recognized, determine the structures of free and bound modules and reveal the residues involved in binding. Our results suggest that peptide stems modulate LysM binding to peptidoglycan. Using these results, we reveal how the LysM module recognizes the GlcNAc-X-GlcNAc motif present in polysaccharides across kingdoms

Study of a Bacillus circulans chitin-binding domain by a green fluorescent protein binding assay and detection of lysozymes by improved zymograms

A fluorescent binding assay was developed to investigate the effects of site-directed mutagenesis on the binding affinity and binding specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. The chitin-binding domain (ChBD) was genetically fused to the N-terminus of the green fluorescent protein, GFP. The polyhistidine-tagged hybrid protein was expressed in Escherichia coli under the dose-dependent regulation of the araBAD promoter and purified using metal affinity-, chitin- or ion-exchange chromatography. Residues suggested to be involved in binding from previous three-dimensional studies were mutated and their contributions to binding and substrate specificity were evaluated by depletion assays. Purified fusion proteins were incubated with chitin beads, polysaccharide-protein complexes were removed by centrifugation and the free protein concentration was measured fluorometrically. The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation. Binding affinity and specificity were alternatively studied by affinity electrophoresis under non-denaturing conditions. Non-conservative substitution of tryptophan residue (W687) with alanine abolished chitin-binding affinity. Double mutation E668K/P689A also impaired binding significantly. Other substitutions in the binding site had little effect on overall affinity for chitin. Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than observed for the wild-type. Furthermore, the ChBD-GFP hybrid protein proved to be useful for specifically labeling cell walls of fungi and yeast and for the detection of fungal infections in tissue samples. Additionally, an improved method for detecting cell lytic activity by a colorbased zymogram was developed. Proteins were separated by electrophoresis in SDS-polyacrylamide gels copolymerized with Remazol-brilliant-blue labeled whole cells of Micrococcus lysodeikticus. After electrophoresis, the enzymes were allowed to refold and lyse the blue-labeled cells embedded in the gel, producing clearing zones in an otherwise bluish gel. This improved zymogram method allows the rapid, sensitive and simultaneous determination of cell lytic specificity, relative activity and molecular weight. This assay should be useful for many research disciplines investigating the role of lysozymes and other cell wall hydrolases capable of refolding after SDS treatment

\u3cem\u3eArabidopsis thaliana\u3c/em\u3e GLX2-1 Contains a Dinuclear Metal Binding Site, but Is Not a Glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV–visible, EPR, and 1H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV–visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in 1H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg246 binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-β-lactamase superfamily

Tracing the molecular and evolutionary determinants of novel functions in protein families

This thesis explores the limits of homology-based inference of protein function and evolution, where overall similarity between sequences can be a poor indicator of functional similarity or evolutionary relationships. Each case presented has undergone different patterns of evolutionary change due to differing selective pressures. Surface adaptations and regulatory (e.g., gene expression) divergence are examined as molecular determinants of novel functions whose patterns are easily missed by assessments of overall sequence similarity. Following this, internal repeats and mosaic sequences are investigated as cases in which key evolutionary events involving fragments of protein sequences are masked by overall comparison. Lastly, virulence factors, which cannot be unified based on sequence, are predicted by analysis of elevated host-mimicry patterns in pathogenic versus non-pathogenic bacterial genomes. These patterns have resulted from unique co-evolutionary pressures that apply to bacterial pathogens, but may be lacking in their close relatives. A recurring theme in the proteins/genes/genomes analyzed is an involvement in microbial pathogenesis or pathogen-defense. Due to the ongoing "evolutionary arms race" between hosts and pathogens, virulence and defense proteins have undergone—and will likely continue to generate—evolutionary novelties. Thus, they demonstrate the necessity to look beyond overall sequence comparison, and assess multiple dimensions of functional innovation in proteins

Expression of functional plant lectins in heterologous systems

The mannose-binding lectin from snowdrop (Galanthus nivalis agglutinin; GNA) was produced in Escherichia coli and purified as a functional protein after denturation/renaturation. Incorporation of the four extra C-terminal residues recently revealed from X-ray crystallographic data demonstrated that these residues increase binding to the glycoprotein carboxypeptidase Y. However, no differences in activities were observed in haemagglutination assays when compared to native GNA and toxicity towards rice brown planthopper (Nilaparvata lugens', BPH) in artificial diet bioassays was unaltered. Site-directed mutagenesis of the carbohydrate-binding site of GNA provided evidence of a direct correlation between the binding potential of GNA to BPH gut glycoprotein 'receptors' and the toxicity levels of GNA towards BPH nymphs. Functional recombinant plant lectins GNA and PHA (Phaseolus vulgaris agglutinin) were expressed in Pichia pastoris using native signal peptides or the Saccharomyces a-factor prepro-sequence to direct secretion. The a-factor prepro-sequence was inefficiently processed unless Glu-Ala repeats were added at the C-terminal end. In the latter case, removal of the Glu-Ala repeats was itself inefficient leading to recombinant lectins with heterogenous N-termini. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed and fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs in Pichia. A fusion protein containing both GNA and GFP (GNA-GFP) was expressed in Pichia pastoris. Simultaneous dual activities (i.e. carbohydrate binding and fluorescence) of recombinant GNA-GFP were demonstrated. Partial cleavage in the linker region resulted in co-purification of GNA which increased the binding activity of the fusion protein. Selective binding of GNA-GFP to haemocytes in the haemolymph of Lacanobia oleracea was observed, both in vitro and when the protein was fed to insects in diet

Structure and function relationships in novel cellulosomal enzymes and cohesindockerin complexes

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e BiomédicasPlant cell walls are the most abundant source of organic carbon on earth, providing an extraordinary supply of energy for various microorganisms. The energetic constrains posed by anaerobic ecosystems lead to the evolution of highly efficient multi-enzymatic complexes, termed cellulosomes, which orchestrate the deconstruction of structural carbohydrates. Clostridium thermocellum cellulosome has been extensively studied as the bacterium exhibits one of the highest growth rates on cellulose. Cellulosomes are assembled by a large non-catalytic multi-modular scaffoldin which contains repeated type I cohesins. Type I dockerin modules, usually located at the C-terminus of enzymes, bind tenaciously to type I cohesins. Scaffoldins may contain a type II dockerin which specifically recognizes type II cohesins located at the cell envelope, allowing the cell surface attachment of cellulosomes. Here a combination of methodologies was applied to study the structure and function relationships of novel cellulosomal enzymes and cohesin-dockerin complexes. Innovative molecular biology and biochemical protocols that can be applied to crystallize and solve the structure of cohesin-dockerin complexes are described in chapter 2. In addition, the crystal structures of two novel type I cohesin-dockerin complexes (CtCohOlpC-Doc124A and CtCohOlpA-Doc918) are described here. They revealed that the two dockerins are unusual since they lack the structural symmetry that supports the dual binding mode typical of type I modules. Thus, these dockerins present a single binding mode and seem to bind preferentially to cohesins located at the bacterium cell surface and not to cellulosomes (chapter 3). Doc124A is the dockerin of CtCel124A, an endoacting cellulase with a superhelical fold that acts in synergy with the major cellulosomal exo-cellulase, Cel48S, during cellulose hydrolysis. The crystal structure of CtCel124A in complex with two cellotriose molecules suggests that the enzyme may target the interface between crystalline and amorphous cellulose (chapter 5). In addition, the structure of a novel type II cohesin-dockerin complex (CtCohScaC2-XDocCipB) was solved. The functional importance of specific dockerin residues was determined. Type II dockerins are suggested to present two different cohesin-binding faces that express different specificities (chapter 4). Finally, the crystal structure of a penta-modular cellulosomal protein (CtXyl5A), previously of unknown function, was assessed (chapter 6). This protein is one of the largest cellulosomal components and comprises a GH5, two CBMs from families 6 and 13, a fibronectin type III-like module, a CBM from family 62 and a type I dockerin. CtGH5 has a canonical (α/β)8-barrel fold and displays specificity for arabinoxylans and as such, is defined as an arabinoxylanase. CtCBM6 adopts a β-sandwich fold and displays affinity for the reaction products generated by CtGH5 and for undecorated xylooligosaccharides. In addition, the penta-modular structure revealed a great flexibility for the CtCBM62 domain.RESUMO - Estrutura e função de novas enzimas celulossomais e de novos complexos coesina-doquerina - A parede celular vegetal constitui uma das principais fontes de carbono do planeta, sendo por isso um extraordinário recurso energético para muitos microrganismos. As limitações energéticas características dos ecossistemas anaeróbios conduziram à evolução de complexos multi-enzimáticos de elevada eficiência, denominados celulossomas, os quais coordenam a degradação dos hidratos de carbono da parede celular vegetal. O Clostridium thermocellum produz um celulossoma relativamente bem caracterizado já que a bactéria apresenta uma das maiores taxas de crescimento em celulose. A organização do celulossoma é efectuada por uma proteína não-catalítica multi-modular. Esta proteína de integração possui uma série de módulos repetidos, denominadas coesinas do tipo I. Na extremidade C-terminal das enzimas celulosomais existem módulos doquerina do tipo I, os quais se ligam fortemente às coesinas do tipo I. As proteínas de integração celulossomal podem conter ainda uma doquerina do tipo II que reconhece especificamente coesinas do tipo II localizadas no envelope celular permitindo, assim, a fixação dos celulossomas à superfície da bactéria. No presente trabalho foram desenvolvidas várias metodologias inovadoras com o intuito de determinar a estrutura e a função de novas enzimas celulossomais e de novos complexos coesina-doquerina. Os protocolos de biologia molecular e bioquímicos aqui descritos (capítulo 2) permitem ultrapassar as dificuldades inerentes à cristalização e, por conseguinte, à resolução da estrutura de complexos coesina-doquerina. Com base nestas metodologias, foram elucidadas as estruturas tridimensionais de dois novos complexos coesina-doquerina do tipo I (CtCohOlpCDoc124A e CtCohOlpA-Doc918). A análise destas estruturas revelou que as suas doquerinas são atípicas, uma vez que não possuem a simetria estrutural característica dos módulos doquerina do tipo I que lhes confere um modo de ligação duplo. Com efeito, estas novas doquerinas apresentam um modo de ligação simples e parecem ligar-se preferencialmente a coesinas localizadas na superfície da bactéria (capítulo 3). A Doc124A é a doquerina da CtCel124A, uma endo-celulase que possui um enrolamento super-helicoidal e que atua em sinergia com a principal exo-celulase celulossomal, a Cel48S, na hidrólise da celulose. A estrutura tridimensional da CtCel124A em complexo com duas moléculas de celotriose sugere que a enzima pode ter como substrato alvo a interface entre as formas cristalina e amorfa da celulose (capítulo 5). Para além da elucidação destas estruturas, foi também resolvida a estrutura de um novo complexo coesina-doquerina do tipo II (CtCohScaC2-XDocCipB). Os resultados sugerem que as doquerinas do tipo II apresentam duas interfaces de ligação a coesinas que expressam diferentes especificidades (capítulo 4). Por último, é apresentada a estrutura tridimensional de uma proteína celulossomal penta-modular (CtXyl5A) e é elucidada a sua função (capítulo 6). Esta proteína é um dos componentes celulossomais de maiores dimensões e compreende uma GH5, dois módulos de ligação a hidratos de carbono (CBMs) das famílias 6 e 13, um módulo de fibronectina do tipo III, um CBM da família 62 e ainda uma doquerina do tipo I. A GH5 apresenta um enrolamento canónico em barril (α/β)8 e possui especificidade para os arabinoxilanos, tendo sido por isso definida como uma arabinoxilanase. O CBM6 apresenta um enrolamento em β-sanduiche e possui afinidade para os produtos de reação gerados pela GH5 e para xilo-oligossacáridos nãodecorados. A estrutura penta-modular desta proteína revelou uma grande flexibilidade no domínio CBM62

Molecular characterization and optimization of enzymes involved in glycosaminoglycan biosynthesis

Glycosaminoglycans are biological active polysaccharides composed of repeating disaccharides composed of a hexuronic acid and a hexosamine. They have various pharmaceutical applications and traditionally this type of molecule is isolated from animal tissue. Since extraction from animal derivatives has serious limitations for the production of a large variety of defined glycosaminoglycans, there is a general interest in developing alternative systems enabling a more tightly controlled synthesis. During this research project we explored alternative ways of controlled chemo-enzymatic synthesis ofmonodisperse and uniform glycosaminoglycans for pharmaceutical applications, with a main focus on heparin. Heparin is a highly sulfated and complex glycosaminoglycan which is worldwide used as an anticoagulant to prevent blood clotting during surgery.Upon closer investigation of the heparin biosynthesis the D-glucuronyl C5-epimerase was recognized as a key enzyme, as this is the only reaction that cannot be done chemically. Two alternative approaches were taken to get closer to an industrial applicable enzyme; improvement of the animal heparin sulfate D-glucuronyl C5-epimerase and identification and isolation of novel candidate C5-epimerases from prokaryotes. Both approaches gave functional production of C5-epimerases. </p