title: Engineering the Pseudomonas aeruginosa II lectin: designing mutants with changed affinity and specificity.
abstract: 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.
authors.affiliation: CEITEC - Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
authors.forename: Zdeněk
authors.initials: Z
authors.lastname: Kříž
authors.forename: Jan
authors.initials: J
authors.lastname: Adam
authors.forename: Jana
authors.initials: J
authors.lastname: Mrázková
authors.forename: Petros
authors.initials: P
authors.lastname: Zotos
authors.forename: Thomais
authors.initials: T
authors.lastname: Chatzipavlou
authors.forename: Michaela
authors.initials: M
authors.lastname: Wimmerová
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Koča
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D003198:Computer Simulation; D017076:Computer-Aided Design; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D018829:Adhesins, Bacterial; D037102:Lectins; C038692:adhesin, Pseudomonas