Papers by Michaela Wimmerova
FEBS Letters, 2003
The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA-IL) in complex with galactose... more The structure of the tetrameric Pseudomonas aeruginosa lectin I (PA-IL) in complex with galactose and calcium was determined at 1.6 A î resolution, and the native protein was solved at 2.4 A î resolution. Each monomer adopts a L L-sandwich fold with ligand binding site at the apex. All galactose hydroxyl groups, except O1, are involved in a hydrogen bond network with the protein and O3 and O4 also participate in the coordination of the calcium ion. The stereochemistry of calcium galactose binding is reminiscent of that observed in some animal C-type lectins. The structure of the complex provides a framework for future design of anti-bacterial compounds.
Nucleic acids research, 2015
Following the discovery of serious errors in the structure of biomacromolecules, structure valida... more Following the discovery of serious errors in the structure of biomacromolecules, structure validation has become a key topic of research, especially for ligands and non-standard residues. ValidatorDB (freely available at http://ncbr.muni.cz/ValidatorDB) offers a new step in this direction, in the form of a database of validation results for all ligands and non-standard residues from the Protein Data Bank (all molecules with seven or more heavy atoms). Model molecules from the wwPDB Chemical Component Dictionary are used as reference during validation. ValidatorDB covers the main aspects of validation of annotation, and additionally introduces several useful validation analyses. The most significant is the classification of chirality errors, allowing the user to distinguish between serious issues and minor inconsistencies. Other such analyses are able to report, for example, completely erroneous ligands, alternate conformations or complete identity with the model molecules. All resul...
The Journal of biological chemistry, Jan 18, 2003
Aleuria aurantia lectin is a fungal protein composed of two identical 312-amino acid subunits tha... more Aleuria aurantia lectin is a fungal protein composed of two identical 312-amino acid subunits that specifically recognizes fucosylated glycans. The crystal structure of the lectin complexed with fucose reveals that each monomer consists of a six-bladed beta-propeller fold and of a small antiparallel two-stranded beta-sheet that plays a role in dimerization. Five fucose residues were located in binding pockets between the adjacent propeller blades. Due to repeats in the amino acid sequence, there are strong similarities between the sites. Oxygen atoms O-3, O-4, and O-5 of fucose are involved in hydrogen bonds with side chains of amino acids conserved in all repeats, whereas O-1 and O-2 interact with a large number of water molecules. The nonpolar face of each fucose residue is stacked against the aromatic ring of a Trp or Tyr amino acid, and the methyl group is located in a highly hydrophobic pocket. Depending on the precise binding site geometry, the alpha- or beta-anomer of the fuc...
Glycobiology, 2014
This study is focused on the analysis and mutagenesis of β-mannosidase from Bacteroides thetaiota... more This study is focused on the analysis and mutagenesis of β-mannosidase from Bacteroides thetaiotaomicron with the aim of broadening its substrate specificity to 2-acetamido-2-deoxy-β-D-mannopyranosyl (β-ManNAc) derivatives. Various conformations ( 4 C 1 , 4 H 5 and 1 S 5 ) of native and modified ligands were docked to the binding site of the protein to determine the most suitable conformation of sugars for further hydrolysis. Key amino acid residues were mutated in silico focusing on stabilizing the acetamido group of β-ManNAc as well as forming the oxazoline intermediate needed for hydrolysis. The results of large set of 5 ns molecular dynamic simulations showed that the majority of the active site residues are involved in substrate interaction and do not exhibit a higher flexibility except for Asn178. Mutations of Asn178 to alanine and Asp199 to serine could lead to a stabilization of the acetamido group in the binding site. So far, in vitro mutagenesis and the screen of a large variety of biological sources were unable to extend β-mannosidase's activity to include β-ManNAc derivatives.
Nucleic Acids Res, 2014
Structure validation has become a major issue in the structural biology community, and an essenti... more Structure validation has become a major issue in the structural biology community, and an essential step is checking the ligand structure. This paper introduces MotiveValidator, a web-based application for the validation of ligands and residues in PDB or PDBx/mmCIF format files provided by the user. Specifically, MotiveValidator is able to evaluate in a straightforward manner whether the ligand or residue being studied has a correct annotation (3-letter code), i.e. if it has the same topology and stereochemistry as the model ligand or residue with this annotation. If not, MotiveValidator explicitly describes the differences. MotiveValidator offers a userfriendly, interactive and platform-independent environment for validating structures obtained by any type of experiment. The results of the validation are presented in both tabular and graphical form, facilitating their interpretation. MotiveValidator can process thousands of ligands or residues in a single validation run that takes no more than a few minutes. MotiveValidator can be used for testing single structures, or the analysis of large sets of ligands or fragments prepared for binding site analysis, docking or virtual screening. MotiveValidator is freely available via the Internet at http://ncbr.muni.cz/ MotiveValidator.
Engineering the Pseudomonas aeruginosa II lectin: designing mutants with changed affinity and specificity
Journal of Computer-Aided Molecular Design, 2014
This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that... more This article focuses on designing mutations of the PA-IIL lectin from Pseudomonas aeruginosa that lead to change in specificity. Following the previous results revealing the importance of the amino acid triad 22-23-24 (so-called specificity-binding loop), saturation in silico mutagenesis was performed, with the intent of finding mutations that increase the lectin's affinity and modify its specificity. For that purpose, a combination of docking, molecular dynamics and binding free energy calculation was used. The combination of methods revealed mutations that changed the performance of the wild-type lectin and its mutants to their preferred partners. The mutation at position 22 resulted in 85% in inactivation of the binding site, and the mutation at 23 did not have strong effects thanks to the side chain being pointed away from the binding site. Molecular dynamics simulations followed by binding free energy calculation were performed on mutants with promising results from docking, and also at those where the amino acid at position 24 was replaced for bulkier or longer polar chain. The key mutants were also prepared in vitro and their binding properties determined by isothermal titration calorimetry. Combination of the used methods proved to be able to predict changes in the lectin performance and helped in explaining the data observed experimentally.
Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients
Nature Structural Biology, 2002
Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the... more Pseudomonas aeruginosa galactose- and fucose-binding lectins (PA-IL and PA-IIL) contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in cystic fibrosis patients. The crystal structure of PA-IIL in complex with fucose reveals a tetrameric structure. Each monomer displays a nine-stranded, antiparallel b-sandwich arrangement and contains two close calcium cations that mediate the binding of fucose in a recognition mode unique among carbohydrate-protein interactions. Experimental binding studies, together with theoretical docking of fucose-containing oligosaccharides, are consistent with the assumption that antigens of the Lewis a (Le(a)) series may be the preferred ligands of this lectin. Precise knowledge of the lectin-binding site should allow a better design of new antibacterial-adhesion prophylactics.
Microbes and Infection, 2004
The opportunistic human pathogen Pseudomonas aeruginosa produces two lectins in close association... more The opportunistic human pathogen Pseudomonas aeruginosa produces two lectins in close association with virulence factors: PA-IL and PA-IIL, which bind to galactose-and fucose/mannose-containing glycoconjugates, respectively. We review here the structural aspects of these lectins relative to their putative roles in host recognition, cell surface adhesion and biofilm formation.
Substrate-Assisted Catalytic Mechanism of O -GlcNAc Transferase Discovered by Quantum Mechanics/Molecular Mechanics Investigation
Journal of the American Chemical Society, 2012
In higher eukaryotes, a variety of proteins are post-translationally modified by adding O-linked ... more In higher eukaryotes, a variety of proteins are post-translationally modified by adding O-linked N-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of O-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer's disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase O-GlcNAc transferase (uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme-substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11,326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S(N)2-like mechanism, in which a nucleophilic attack by O(Ser), facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1-O(Ser) = 1.92 Å and C1-O1 = 3.11 Å. The activation energy for this passage was estimated to be ~20 kcal mol(-1). These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the N-acetamino group of the donor participates in the catalytic mechanism.
Chemistry - A European Journal, 2013
Chemistry - A European Journal, 2011
lated scaffold 10 [58] (Scheme 2). The acetylated glycoconjugate 11 was then obtained by conjugat... more lated scaffold 10 [58] (Scheme 2). The acetylated glycoconjugate 11 was then obtained by conjugation with the carbohydrate derivative A and unmasking of the hydroxyl groups gave the desired glycocluster 12. As attested by the
Bioinformatics, 2008
The new version of the TRITON program provides userfriendly graphical tools for modeling protein ... more The new version of the TRITON program provides userfriendly graphical tools for modeling protein mutants using the external program MODELLER and for docking ligands into the mutants using the external program AutoDock. TRITON can now be used to design ligand-binding proteins, to study protein-ligand binding mechanisms or simply to dock any ligand to a protein.
ChemInform Abstract: In silico Mutagenesis and Docking Studies of Pseudomonas aeruginosa PA-IIL Lectin - Predicting Binding Modes and Energies
ChemInform, 2009
Structural insights into Aspergillus fumigatus lectin specificity: AFL binding sites are functionally non-equivalent
Acta crystallographica. Section D, Biological crystallography, 2015
The Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin fam... more The Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological...
PLoS ONE, 2013
Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many othe... more Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, Le Y being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,Lselenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus' conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection. Citation: Houser J, Komarek J, Kostlanova N, Cioci G, Varrot A, et al. (2013) A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia -Structure, Specificity and Possible Role in Fungal Pathogenicity. PLoS ONE 8(12): e83077.
J Struct Biol, 2014
The crystal structure of the N-terminal domain of the RNA polymerase d subunit (Nd) from Bacillus... more The crystal structure of the N-terminal domain of the RNA polymerase d subunit (Nd) from Bacillus subtilis solved at a resolution of 2.0 Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important b sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nd, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni 2+ ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.
Proteins, Jan 15, 2005
PA-IIL is a fucose-binding lectin from Pseudomonas aeruginosa that is closely related to the viru... more PA-IIL is a fucose-binding lectin from Pseudomonas aeruginosa that is closely related to the virulence factors of the bacterium. Previous structural studies have revealed a new carbohydrate-binding mode with direct involvement of two calcium ions (Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Perez S, Wu AM, Gilboa-Garber N, Imberty A. Structural basis for selective recognition of oligosaccharides from cystic fibrosis patients by the lectin PA-IIL of Pseudomonas aeruginosa. Nat Struct Biol 2002;9:918-921). A combination of thermodynamic, structural, and computational methods has been used to study the basis of the high affinity for the monosaccharide ligand. A titration microcalorimetry study indicated that the high affinity is enthalpy driven. The crystal structure of the tetrameric PA-IIL in complex with fucose and calcium was refined to 1.0 A resolution and, in combination with modeling, allowed a proposal to be made for the hydrogen-bond network in the binding site...
Uploads
Papers by Michaela Wimmerova