Refined Crystallographic Structure of Pseudomonas aeruginosa Exotoxin A and its Implications for the Molecular Mechanism of Toxicity Joseph E. Wedekind 1 , Christine B. Trame 1 , Magdalena Dorywalska 1 Patrice Koehl 1 , Tanya M. Raschke 1 , Marian McKee 2 , David FitzGerald 2 R. John Collier 3 and David B. McKay 1 * 1 Department of Structural Biology, Stanford University School of Medicine, Stanford CA 94305, USA 2 Laboratory of Molecular Biology, Division of Cancer Biology, National Cancer Institute, National Institutes of Health, Bethesda MD 20892, USA 3 Department of Microbiology and Molecular Genetics Harvard Medical School Boston, MA 02115, USA Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to speci®c cell surface receptors and intracellular traf®cking 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 Ê 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 trans- ferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, re®ned with data to 1.62 A Ê and 1.45 A Ê resolution, respectively. The re®ned 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 signi®cantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/¯exibility at the site, rather than to differences in static tertiary structure. Comparison of the re®ned 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 cat- alytic domain and Glu348 of domain II that restrains the substrate bind- ing cleft in a conformation that precludes NAD  binding. The re®ned structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication. # 2001 Academic Press Keywords: exotoxin; Pseudomonas aeruginosa toxicity; crystal structure; furin; pH-induced conformational change *Corresponding author Introduction Exotoxin A of Pseudomonas aeruginosa (PE) 1 is a virulence factor secreted in response to environ- mental iron scarcity. 2 The mature protein has 613 amino acid residues and four disul®de linkages. PE is highly toxic to eukaryotic cells, causing the arrest of protein synthesis. This is accomplished Present address; J. E. Wedekind, Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL 60064, USA. Abbreviations used: PE, Pseudomonas aeruginosa exotoxin A; ER, endoplasmic reticulum; PEG, polyethylene glycol; CCD, charge-coupled device; SSRL, the Stanford Synchrotron Radiation Laboratory; a calc , calculated phases; eEF-2, eukaryotic elongation factor 2; DT, diphtheria toxin. E-mail address of the corresponding author: Dave.McKay@Stanford.edu doi:10.1006/jmbi.2001.5195 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 314, 823±837 0022-2836/01/040823±15 $35.00/0 # 2001 Academic Press