title: Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity.
abstract: Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Wedekind
authors.forename: C B
authors.initials: CB
authors.lastname: Trame
authors.forename: M
authors.initials: M
authors.lastname: Dorywalska
authors.forename: P
authors.initials: P
authors.lastname: Koehl
authors.forename: T M
authors.initials: TM
authors.lastname: Raschke
authors.forename: M
authors.initials: M
authors.lastname: McKee
authors.forename: D
authors.initials: D
authors.lastname: FitzGerald
authors.forename: R J
authors.initials: RJ
authors.lastname: Collier
authors.forename: D B
authors.initials: DB
authors.lastname: McKay
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001665:Binding Sites; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D045683:Furin; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018583:Pliability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D045683:Furin; D008024:Ligands; D013381:Subtilisins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A