基于单分子方法解析IgM与C1q及MBL动态相互作用的物理基础

IgM is an important antibody of the primary immune response, playing an essential role in the first line of defense against foreign pathogens. However, it also plays a significant role in the pathogenesis of several autoimmune diseases, including ischaemia-reperfusion (I/R) injury (during, for example, heart attack and stroke) and systemic lupus erythematosus (SLE). In these, tissue inflammation and injury are believed to be at least partly owing to deleterious complement activities resulting from interactions of MBL (in I/R injury) and complement C1q (in SLE) with IgM auto-antibodies. However, although the role of these molecules in these diseases has been reasonably well established, the underlying molecular details have yet to be adequately described. Clearly, a better understanding of the physical mechanisms by which IgM interacts with these activating components of complement is essential for the development of effective treatments for these debilitating illnesses, for which there are presently none. The working atomic model of human IgM recently determined by our group has provided a basic physical structure with which to understand major aspects of its functioning. In this proposal, building on this model, we plan to provide a structural and energetic understanding of the interaction between C1q and MBL with IgM, information that is essential for the development of effective therapeutics for I/R injury and SLE. Specifically, we will combine molecular dynamics simulations and multifaceted single molecule structural and force spectroscopic approaches to unravel the structural and energetic details of the IgM/C1q and IgM/MBL complexes. Molecular dynamics simulations will provide atomic-level structural information as well as quantitative predictions of the interactions that can be verified or subsequently improved with targeted experiments, employing single molecule AFM imaging, single molecule FRET, and multiplex force spectroscopy using our home-built electromagnetic tweezers apparatus, as well as investigations of select mutants of the C1q and MBL globular domains. Such detailed physical information of these interactions will enable the future design, aided by molecular dynamics simulations, of small molecule modulators effective for I/R injury and SLE.

免疫球蛋白M（IgM）是多种自身免疫疾病（如：心肌梗死和中风过程中的缺血再灌注(I/R)损伤和系统性红斑狼疮（SLE））中的重要因素之一。补体系统的激活是这类严重疾病的重要原因之一。IgM/MBL和IgM/C1q的相互作用是I/R和SLE致病的关键步骤，但对其分子机制却知之甚少，严重阻碍了有效药物或治疗方法的发展。我们实验室结合单分子成像与分子模拟技术，已成功构建了人源IgM的原子结构模型。以此为基础，我们在本项目中进一步应用分子动力学模拟，结合单分子结构分析（FM-AFM，FRET）与单分子力谱测量，系统研究IgM与C1q及IgM与MBL相互作用的结构基础，构建不同条件下的自由能景观（free energy landscape）。通过对这些重要分子组合的物理机制进行全面解析，建立完整的与核心氨基酸残基相关联的分子模型，并在此基础上探索调控这些相互作用的可能性，为未来开发相关药物做出贡献。

本项目的总体目标为通过理论模拟与单分子等实验手段研究免疫球蛋白M（IgM）多聚体与补体分子C1q的相互作用。针对上述目标，本项目:（1）首先通过多种条件下的分子动力学（MD）模拟计算，优化完善了IgM以及C1q的结构模型；（2）通过发展抗原多肽-IgM结合的实验体系，应用生物膜层干涉等技术，解析了IgM多聚体与抗原结合、以及与C1q结合位点相互作用过程中的构象动力学变化，建立了其自由能景观描述；（3）在此基础上发展研究IgM与其结合蛋白作用的分子动力学计算方法，并应用自主研制的低温原子力显微镜等独特的实验手段初步建立了IgM与C1q相互作用的结构模型。为进一步研究IgM与C1q相互作用，探索免疫激活经典途径的关键步骤奠定了分子结构基础，也为系统性红斑狼疮（SLE）等（IgM与C1q相互作用）相关疾病发病机制的探索与治疗提供了技术手段。