A model of anthrax toxin lethal factor bound to protective antigen.

Anthrax toxin is made up of three proteins: the edema factor (EF), lethal factor (LF) enzymes, and the multifunctional protective antigen (PA). Proteolytically activated PA heptamerizes, binds the EF/LF enzymes, and forms a pore that allows for EF/LF passage into host cells. Using directed mutagenesis, we identified three LF-PA contact points defined by a specific disulfide crosslink and two pairs of complementary charge-reversal mutations. These contact points were consistent with the lowest energy LF-PA complex found by using Rosetta protein-protein docking. These results illustrate how biochemical and computational methods can be combined to produce reliable models of large complexes. The model shows that EF and LF bind through a highly electrostatic interface, with their flexible N-terminal region positioned at the entrance of the heptameric PA pore and thus poised to initiate translocation in an N- to C-terminal direction.

[1]  J. Florián,et al.  Calcium‐independent calmodulin binding and two‐metal–ion catalytic mechanism of anthrax edema factor , 2005, The EMBO journal.

[2]  S. Leppla,et al.  Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[3]  D. Lacy,et al.  Mapping the Anthrax Protective Antigen Binding Site on the Lethal and Edema Factors* , 2002, The Journal of Biological Chemistry.

[4]  R. Collier,et al.  A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen. , 2000, Biochemistry.

[5]  R. Collier,et al.  Effect of Anthrax Toxin's Lethal Factor on Ion Channels Formed by the Protective Antigen (*) , 1995, The Journal of Biological Chemistry.

[6]  R. Collier,et al.  Identification of residues lining the anthrax protective antigen channel. , 1998, Biochemistry.

[7]  S. Harrison,et al.  Structure of heptameric protective antigen bound to an anthrax toxin receptor: a role for receptor in pH-dependent pore formation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  D. Lacy,et al.  The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Lacy,et al.  Binding Stoichiometry and Kinetics of the Interaction of a Human Anthrax Toxin Receptor, CMG2, with Protective Antigen* , 2004, Journal of Biological Chemistry.

[10]  K D Paull,et al.  Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. , 1998, Science.

[11]  K. Klimpel,et al.  Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. , 1992, The Journal of biological chemistry.

[12]  Jadwiga Bienkowska,et al.  Crystal structure of the anthrax lethal factor , 2001, Nature.

[13]  R. Collier,et al.  Characterization of membrane translocation by anthrax protective antigen. , 1998, Biochemistry.

[14]  M. Mock,et al.  Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. , 1998, Biochemical and biophysical research communications.

[15]  Jeffrey J. Gray,et al.  Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. , 2003, Journal of molecular biology.

[16]  J S Wall,et al.  Anthrax protective antigen forms oligomers during intoxication of mammalian cells. , 1994, The Journal of biological chemistry.

[17]  K. Christensen,et al.  Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin. , 2004, Journal of molecular biology.

[18]  R. Collier,et al.  Stoichiometry of anthrax toxin complexes. , 2002, Biochemistry.

[19]  E. Hewlett,et al.  Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin , 1988, Infection and immunity.

[20]  R. Collier,et al.  Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[21]  O. Schueler‐Furman,et al.  Progress in protein–protein docking: Atomic resolution predictions in the CAPRI experiment using RosettaDock with an improved treatment of side‐chain flexibility , 2005, Proteins.

[22]  R. Collier,et al.  Protein translocation through anthrax toxin channels formed in planar lipid bilayers. , 2004, Biophysical journal.

[23]  D. Lacy,et al.  Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen , 2002, Proceedings of the National Academy of Sciences of the United States of America.