MD Study of Anthrax Edema Factor:Calmodulin Complexes

描述(由申请人提供)：该项目的长期目标是了解钙调蛋白(CAM)与炭疽水肿因子(EF)有效结合以及产生一个功能齐全的腺酰环化酶活性部位的精确构象和动态要求。更详细地了解水肿因子、钙调蛋白和底物类似物之间的复合体的结构和动力学，应有助于今后设计治疗炭疽感染的基于结构的药物。本申请建议使用分子动力学模拟(MD)方法研究大量包含EF单点突变的EF-CaM复合体，这些突变已知会影响CaM结合或腺苷环化酶催化活性。这项建议的具体目的是：1)表征EF突变的构象和动态后果，这些突变导致CaM与EF结合的已知缺陷。将生成EF：CaM界面上包含EF突变的EF-CAM复合体的MD模拟输入文件，这些文件已知会影响CaM结合或CaM诱导的腺苷环化酶活性(L523A、K25A、Q526A、V529A和D647A)，然后使用Insight II软件进行MD模拟。随后的分析将集中在野生型和EF变异复合体之间MD轨迹的构象和动力学差异。MD结果将与这些EF变体被CaM激活的能力的动力学数据相关联，以确定CaM与EF最有效结合的要求。2)表征EF突变对EF腺酰环化酶活性产生不利影响的构象和动态后果。将生成在EF腺酰环化酶活性部位含有EF突变的EF：CaM复合体的MD模拟输入文件，并随后进行MD模拟，并且随后进行MD模拟。将在没有和存在结合底物类似物3‘-脱氧-ATP的情况下进行MD模拟。对所产生的ME)轨迹的分析将集中于野生型和EF变异复合体之间的构象和动力学差异。所有结果都将与这些EF变体被CaM激活的能力的现有酶动力学数据相关联，以确定开发全功能EF腺酰环化酶的精确要求。

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.