Thermodynamic and structural studies on the formation of the C3 convertase

C3转化酶形成的热力学和结构研究

• 批准号: 7319517
• 负责人: JOHN D LAMBRIS
• 金额: $ 40.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7319517
• 关键词: Affinity; Alternative Complement Pathway; Amides; Binding; Biochemical; Biochemical Reaction; Buffers; C3bi; Calorimetry; Catalytic Domain; Cleaved cell; Complement; Complement 3 Convertase; Complement 3b; Complement Activation; Complement Factor B; Complement Factor D; Complex; Computing Methodologies; Coupled; Crystallization; Crystallography; Dependence; Deuterium; Entropy; Ethanolamines; Free Energy; Glycerol; Goals; Grant; Heating; Human; Hydrogen; Hydrolysis; Individual; Kinetics; Lectin; Ligands; Liquid substance; Maps; Mass Spectrum Analysis; Measurement; Measures; Medical; Methods; Molecular; Molecular Conformation; Multienzyme Complexes; Pathway interactions; Phase; Physiological; Play; Principal Investigator; Process; Proteins; Rate; Research Design; Resolution; Role; Site; Site-Directed Mutagenesis; Sodium Chloride; Solutions; Solvents; Specificity; Structure; Surface; Surface Plasmon Resonance; System; Temperature; Therapeutic Intervention; Thermodynamics; Time; Titrations; alternative pathway complement C3 convertase; base; conformational conversion; design; enthalpy; ethanolamine; inhibitor/antagonist; insight; ionization; programs; research study; sensor; three dimensional structure

DESCRIPTION (provided by applicant): Among the complement pathways, the alternative pathway plays a predominant role in amplification of effector functions in the complement system and has been considered a key target for complement therapeutic interventions. Its activation is initiated when factor B binds to "C3b-like C3" (C3(H2O)). Factor D then cleaves factor B into Ba and Bb and the resulting complex, C3(H20)Bb, serves as the initial C3 convertase capable of cleaving C3 to C3b. The catalytic subunit of the enzyme complex is located in the Bb fragment. The newly generated C3b, as well as any C3b that is generated through the activation of the classical or lectin pathway, can bind another molecule of B and, after factor D cleavage, the alternative pathway C3bBb convertase is established. Although biochemical details of the interaction between C3 and factor B have been accumulated over the past three decades, a more detailed characterization of this binding process is essential for the understanding of its physiological relevance and its implications for C3 convertase formation and function. The long-term goal of this proposal is to identify the structural determinants associated with the formation of C3 convertase. The proposed study will involve a detailed structural and functional analysis associated with the formation of the alternative pathway C3 convertase, by studying the interaction between the human C3(H2O), C3b and the complement proteins factor B and its fragments, Bb and Ba. Protein crystallography, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and hydrogen exchange/mass spectrometry (HDX-MS) will be used to assess the molecular forces that impart specificity and recognition to the interactions between C3 and factor B. The functional sites of the interacting molecules will be mapped using HDX-MS, crystallization, computational methods, and site-directed mutagenesis. The proposed studies are designed to provide basic information on the structural features of CS-factor B interactions as they relate to complement C3 convertase functions. These studies should provide insight into how the complement C3 convertase is formed and also assist in the design of specific inhibitors that may have important medical applications.

描述（由申请人提供）：在补体途径中，替代途径在补体系统效应器功能的放大中发挥着主导作用，并被认为是补体治疗干预的关键靶点。当因子B与“C3 b样C3”（C3（H2O））结合时，其激活开始。然后，因子D将因子B切割成Ba和B B b，并且所得复合物C3（H2O）B B b用作能够将C3切割成C3 B b的初始C3转化酶。酶复合物的催化亚基位于Bb片段中。新产生的C3 B以及通过经典途径或凝集素途径活化产生的任何C3 B可以结合B的另一分子，并且在因子D裂解后，建立了替代途径C3 bB B b转化酶。虽然在过去的三十年中已经积累了C3和因子B之间的相互作用的生物化学细节，更详细的表征这种结合过程是必不可少的了解其生理相关性和C3转化酶的形成和功能的影响。该提案的长期目标是确定与C3转化酶形成相关的结构决定因素。拟议的研究将涉及与旁路途径C3转化酶形成相关的详细结构和功能分析，通过研究人C3（H2O），C3 B和补体蛋白因子B及其片段，B B和Ba之间的相互作用。将使用蛋白质晶体学、等温滴定量热法（ITC）、表面等离子体共振（SPR）和氢交换/质谱法（HDX-MS）评估赋予C3和因子B之间相互作用特异性和识别的分子力。相互作用分子的功能位点将使用HDX-MS、结晶、计算方法和定点诱变进行作图。拟议的研究旨在提供CS-因子B相互作用的结构特征的基本信息，因为它们与补体C3转化酶功能有关。这些研究应该提供深入了解补体C3转化酶是如何形成的，也有助于设计可能具有重要医学应用的特异性抑制剂。