title: Mechanism of reactant and product dissociation from the anthrax edema factor: a locally enhanced sampling and steered molecular dynamics study.
abstract: The anthrax edema factor is a toxin overproducing damaging levels of cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) from ATP. Here, mechanisms of dissociation of ATP and products (cAMP, PPi) from the active site are studied using locally enhanced sampling (LES) and steered molecular dynamics simulations. Various substrate conformations and ionic binding modes found in crystallographic structures are considered. LES simulations show that PPi and cAMP dissociate through different solvent accessible channels, while ATP dissociation requires significant active site exposure to solvent. The ionic content of the active site directly affects the dissociation of ATP and products. Only one ion dissociates along with ATP in the two-Mg(2+) binding site, suggesting that the other ion binds EF prior to ATP association. Dissociation of reaction products cAMP and PPi is impaired by direct electrostatic interactions between products and Mg(2+) ions. This provides an explanation for the inhibitory effect of high Mg(2+) concentrations on EF enzymatic activity. Breaking of electrostatic interactions is dependent on a competitive binding of water molecules to the ions, and thus on the solvent accessibility of the active site. Consequently, product dissociation seems to be a two-step process. First, ligands are progressively solvated while preserving the most important electrostatic interactions, in a process that is dependent on the flexibility of the active site. Second, breakage of the electrostatic bonds follows, and ligands diffuse into solvent. In agreement with this mechanism, product protonation facilitates dissociation.
authors.affiliation: Unité de Bioinformatique Structurale, URA CNRS 2185, Institut Pasteur, 25, rue du Dr Roux, F-75015 Paris, France. leandro@ifsc.usp.br
authors.forename: Leandro
authors.initials: L
authors.lastname: Martínez
authors.forename: Thérèse E
authors.initials: TE
authors.lastname: Malliavin
authors.forename: Arnaud
authors.initials: A
authors.lastname: Blondel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001408:Bacillus anthracis / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D008274:Magnesium