Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility
نویسندگان
چکیده
Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.
منابع مشابه
Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1
At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the b...
متن کاملElucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.
Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 k...
متن کاملAntimicrobial Photothermal Treatment of Pseudomonas Aeruginosa by a Carbon Nanoparticles-Polypyrrole Nanocomposite
Background: Nowadays, it is needed to explore new routes to treat infectious bacterial pathogens due to prevalence of antibiotic-resistant bacteria. Antimicrobial photothermal therapy (PTT), as a new strategy, eradicates pathogenic bacteria.Objective: In this study, the antimicrobial effects of a carbon nanoparticles-polypyrrole nanocomposite (C-PPy) upon laser irradiation were investigat...
متن کاملCloning, Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections
Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alt...
متن کامل