Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity

J Mol Biol. 2001 Dec 7;314(4):823-37. doi: 10.1006/jmbi.2001.5195.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ADP Ribose Transferases*
  • Amino Acid Sequence
  • Bacterial Toxins*
  • Binding Sites
  • Computer Simulation
  • Crystallization
  • Crystallography, X-Ray
  • Exotoxins / chemistry*
  • Exotoxins / genetics
  • Exotoxins / metabolism
  • Exotoxins / toxicity*
  • Furin
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Ligands
  • Models, Molecular
  • Mutation / genetics
  • Pliability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Pseudomonas aeruginosa / chemistry*
  • Pseudomonas aeruginosa / enzymology
  • Pseudomonas aeruginosa / genetics
  • Sequence Alignment
  • Static Electricity
  • Structure-Activity Relationship
  • Subtilisins / metabolism
  • Virulence Factors*

Substances

  • Bacterial Toxins
  • Exotoxins
  • Ligands
  • Virulence Factors
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa
  • Subtilisins
  • Furin

Associated data

  • PDB/1IKP
  • PDB/1IKQ