孤独症相关蛋白ANKS1B Ankyrin Repeats结构域及其蛋白质复合体的晶体结构研究

ANKS1B, Ankyrin repeat and sterile alpha motif domain containing protein 1B, is a tyrosine kinase signal transduction gene that is primarily expressed in the brain and testis. ANKS1B is one of major component of postsynaptic region, playing critical roles in many developmental processes such as neurogenesis, neuronal circuit formation and synaptic plasticity. Not surprisingly, mutations of ANKS1B are known to cause many forms of diseases including Autism, Schizophrenia and cancers. However, the biological mechanism of ANKS1B remain largely elusive. ANKS1B contains multiple ankyrin repeats near its N-terminus as well as two SAM domains and, in the C-terminal half of the protein, a PTB (PID) domain, which belongs to the Dab-like subtype of PTBs. These domains are connected by an intermediate region with no obvious structural elements, which harbours the majority of currently known phosphorylation sites. Many protein associated in Genome-wide association studies have been repeatedly reported, namely LRP and GNB, but if and how they regulate ANKS1B function, or which protein is responsible for which site, remains unclear. Little is known about protein structure and interaction, especially the N-terminal Ankyrin Repeats domain..To reveal the structure and interaction groups of Ankyrin Repeats domain of ANKS1B, which mainly charged for protein interactions, is the feasible way to figure out the important role of ANKS1B. Our previous studies had obtained excellent crystallographic data on multi-Ankyrin Repeats domain, especially the structures of 24 Ankyrin Repeats of AnkyrinB and 10 Ankyrin Repeats of Espin1. Here we explored the highly resolution structure of ANKS1B Ankyrin Repeats domain. We employ the structural biology techniques to analyze the structure of Ankyrin Repeats, set to apply affinity purification coupled with LC-MS/MS and yeast two-hybrid assay to find out the interactome. The X-ray crystallographic studies of ANKS1B and interaction proteins complex, together with biochemical and cell biology experiments, not only revealed the exquisite residues governing Ankyrin Repeats domain interactions but also allowed us to identify Ranbp5 and RGS6 as the new ANKS1B binding partners. Finally, these structures can explain numerous ANKS1B mutations identified in patients suffering from Autism, schizophrenia, cancers and other diseases. We propose that the ANKS1B mutations found in patients that fall into the above structures will have higher chance to be disease-relevant mutations and thus are given higher priority to be investigated. With these structure studies, we will be able to decipher the theoretical basis for the etiological study of Autism and other diseases. The results shall shed light on the pathogenesis of Autism in human patients and lays the foundation for the design of specific pharmaceutical agents against the disease.

ANKS1B作为突触后膜的主要蛋白质组分，在神经系统的发育发生中起着重要作用。ANKS1B功能异常与孤独症等多种疾病密切相关，但与功能对应的分子机制研究甚少。在ANKS1B这一多结构域蛋白质中，Ankyrin repeats结构域是其与蛋白质互作的主要结构域，研究其晶体结构和相互作用组，是揭示ANKS1B在神经突触中重要作用的可行之路。本项目在既往研究的基础上，从ANKS1B Ankyrin Repeats结构域的晶体结构入手，通过GST-pull down结合LC-MS/MS和酵母双杂两种方法寻找互作蛋白，解析蛋白质复合体晶体结构。通过对复合体的晶体结构研究，明确以ANKS1B为核心的蛋白质复合体在突触后膜发挥生物学功能的分子机制，揭示相互作用的关键位点。结合临床病人中出现的基因突变位点，分析破坏蛋白质复合体的影响。为孤独症等疾病的病因学研究提供理论依据，也为特异性靶向药物设计奠定基础。

在本项目中，我们利用生物化学与结构生物学的技术手段，解析了ANKS1B Ankyrin Repeats结构域的分子结构，得到了1.9Å晶体结构，前一半研究目标已圆满完成；通过多种方法寻找ANKS1B Ankyrin Repeats结构域互作蛋白并进行验证。然而在我们的表达体系中，始终没有得到均一稳定的蛋白质复合体。因此，本项目后期的研究内容进行了调整。我们对与ANKS1B类似的突触支架蛋白CASK进行了结构生物学研究。CASK作为突触中重要的组成蛋白，其基因突变明确与多种神经疾病相关。研究CASK蛋白也可以为研究神经系统疾病提供理论依据。因此，我们通过多种实验手段验证了CASK与互作蛋白Caskin1的相互作用，解析了CASK_CAMK/Caskin1_CID蛋白质复合体的晶体结构。通过晶体结构和序列比对分析，我们找到了CASK_CAMK互作基序CID的基本特征。在蛋白质数据库中，利用我们找到的互作基序特点，新发现了CASK_CAMK潜在的四种互作蛋白。其中CIC和LRRK1参与神经发育过程，多种CID蛋白可能在不同的时间，空间与CASK结合，从而实现神经发育的阶段性功能。综合生化实验结果和晶体结构分析，我们系统总结分析CASK_CAMK互作的三种模式，讨论了CASK做了重要的支架蛋白，其互作组成的蛋白质网络对于神经功能的重要性。