Thermodynamic and structural studies on the formation of the C3 convertase

描述(申请人提供)：在补体途径中，替代途径在增强补体系统的效应器功能方面发挥着主要作用，并被认为是补体治疗干预的关键靶点。当B因子与“C3b样C3”(C3(H2O))结合时，它的激活被启动。然后，D因子将B因子裂解成BA和BB，生成的复合体C3(H20)BB，作为能够将C3裂解成C3b的初始C3转换酶。酶复合体的催化亚基位于BB片段。新产生的C3b，以及通过激活经典或凝集素途径产生的任何C3b，都可以与另一个B分子结合，在D因子切割后，建立了替代途径C3bBb转换酶。虽然C3和B因子相互作用的生化细节在过去30年中已经积累，但更详细地描述这种结合过程对于理解它的生理相关性及其对C3转换酶的形成和功能的影响是至关重要的。这项提议的长期目标是确定与C3转换酶形成相关的结构决定因素。这项拟议的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，涉及与替代途径C3转换酶的形成有关的详细结构和功能分析。蛋白质结晶学、等温滴定量热法(ITC)、表面等离子体共振(SPR)和氢交换/质谱仪(HDX-MS)将用于评估给予C3和B因子相互作用的特异性和识别性的分子作用力。相互作用分子的功能位点将使用HDX-MS、结晶、计算方法和定点突变来绘制。拟议的研究旨在提供关于CS-B因子相互作用的结构特征的基本信息，因为它们与补体C3转换酶功能有关。这些研究应该有助于深入了解补体C3转换酶是如何形成的，并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.