MD Study of Anthrax Edema Factor:Calmodulin Complexes

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DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the precise conformational and dynamic requirements for both effective binding of calmodulin (CAM) to the anthrax edema factor (EF) and the generation of a fully functional adenylyl cyclase active site. A more detailed understanding of the structure and dynamics of the complex between the edema factor, calmodulin and substrate analogs should facilitate future efforts at designing structure-based drugs for the treatment of anthrax infections. This application proposes to investigate, using the molecular dynamics simulation (MD) approach, a large number of EF-CaM complexes containing single-site mutations in EF which are already known to affect either CaM binding or adenylyl cyclase catalytic activity. The specific aims of this proposal are to: 1) Characterize the conformational and dynamic consequences of EF mutations which give rise to known defects in CaM binding to EF. MD simulation input files for EF-CAM complexes containing EF mutations, at the EF:CaM interface, known to affect either CaM binding or CaM-induced adenylyl cyclase activity (L523A, K25A, Q526A, V529A and D647A), will be generated, and then subjected to MD simulations using Insight II software. The subsequent analysis will focus on conformational and dynamic differences in the MD trajectories between wild-type and EF variant complexes. MD results will be correlated with kinetic data on the ability of these EF variants to be activated by CaM, in order to determine the requirements for most effective binding of CaM to EF. 2) Characterize the conformational and dynamic consequences of EF mutations which adversely affect EF adenylyl cyclase activity. MD simulation input files for EF:CaM complexes containing EF mutations, at the EF adenylyl cydase active site, known to affect the CaM-induced adenylyl cyclase activity (K346R, K353R, K353A, H577N, H577D, N583A, N583Q, N583H, E588A, D50A and N639A) will be generated, and subsequently subjected to MD simulations. MD simulations will be performed, both in the absence and presence of bound substrate analog 3'-deoxy-ATP. Analysis of the resulting ME) trajectories will focus on the conformational and dynamic differences between the wild-type and EF variant complexes. All results will be correlated with available enzyme kinetic data on the ability of these EF variants to be activated by CaM, to determine the precise requirements for the development of a fully-functional EF adenylyl cyclase.