Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery.

• MeSH
• databáze proteinů * MeSH
• internet MeSH
• konformace proteinů MeSH
• lektiny chemie   MeSH
• makromolekulární látky chemie   MeSH
• molekulární konformace * MeSH
• molekulární modely MeSH
• software * MeSH
• vazebná místa MeSH
• zinkové prsty MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• LecB protein, Pseudomonas aeruginosa MeSH Prohlížeč
• lektiny MeSH
• makromolekulární látky MeSH

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.

• MeSH
• bakteriální adheziny chemie   genetika   metabolismus   MeSH
• design s pomocí počítače MeSH
• lektiny chemie   genetika   metabolismus   MeSH
• metabolismus sacharidů MeSH
• mutace MeSH
• mutageneze * MeSH
• počítačová simulace MeSH
• Pseudomonas aeruginosa chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• termodynamika MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adhesin, Pseudomonas MeSH Prohlížeč
• bakteriální adheziny MeSH
• lektiny MeSH

The effect of terminal GLY114* deletion on the binding affinity of the PA-IIL lectin toward L: -fucose was investigated. Both experimental (isothermal titration calorimetry) and computational (molecular dynamics simulations) methods have shown that the deletion mutation decreases the L-fucose affinity. It implies that the PA-IIL saccharide binding affinity is influenced by the dimerization of the lectin. A detailed analysis of computational data confirms the key role of electrostatic interactions in the PA-IIL/saccharide binding.

• MeSH
• bakteriální adheziny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• fukosa chemie   metabolismus   MeSH
• kalorimetrie metody   MeSH
• kinetika MeSH
• kompetitivní vazba MeSH
• krystalizace MeSH
• kvarterní struktura proteinů MeSH
• lektiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• mutace * MeSH
• počítačová simulace MeSH
• Pseudomonas aeruginosa genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sekvenční delece MeSH
• terciární struktura proteinů MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adhesin, Pseudomonas MeSH Prohlížeč
• bakteriální adheziny MeSH
• fukosa MeSH
• lektiny MeSH
• rekombinantní proteiny MeSH

BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.

• MeSH
• bakteriální adheziny chemie   genetika   MeSH
• chromatografie afinitní MeSH
• jednonukleotidový polymorfismus MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lektiny chemie   genetika   MeSH
• molekulární modely MeSH
• monosacharidy chemie   MeSH
• mutageneze cílená MeSH
• proteinové inženýrství MeSH
• Pseudomonas aeruginosa genetika   MeSH
• Ralstonia solanacearum chemie   MeSH
• rekombinantní proteiny chemie   izolace a purifikace   MeSH
• rostlinné lektiny chemie   MeSH
• substituce aminokyselin MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adhesin, Pseudomonas MeSH Prohlížeč
• bakteriální adheziny MeSH
• lektiny MeSH
• monosacharidy MeSH
• rekombinantní proteiny MeSH
• rostlinné lektiny MeSH