Thermodynamic and structural studies on the formation of the C3 convertase

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功能的扩增中起主导作用，被认为是补体治疗干预的关键靶点。当因子B与“c3b样C3”（C3(H2O)）结合时，它的激活被启动。然后，因子D将因子B裂解为Ba和Bb，生成的复合物C3(H20)Bb作为C3转化酶，能够将C3裂解为C3b。酶复合物的催化亚基位于Bb片段中。新生成的C3b，以及通过经典途径或凝集素途径激活产生的任何C3b，都可以结合另一个B分子，并在因子D切割后，建立替代途径C3bBb转化酶。虽然C3和因子B之间相互作用的生化细节在过去的三十年中已经积累，但对这一结合过程的更详细的表征对于理解其生理相关性及其对C3转化酶形成和功能的影响至关重要。本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。该研究将通过研究人类C3（H2O）、C3b与补体蛋白因子B及其片段Bb和Ba之间的相互作用，对与替代途径C3转化酶形成相关的详细结构和功能进行分析。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）将用于评估赋予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来绘制。拟议的研究旨在提供有关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。这些研究应该为补体C3转化酶如何形成提供深入的见解，也有助于设计可能具有重要医学应用的特定抑制剂。

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.