DOMAIN SPECIFIC INTERACTIONS OF CENTRIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。中心体是微管组织中心（MTOC）的重要组成部分，在有丝分裂过程中介导染色体分离。除了与其他MTOC组分相互作用外，已知它还与细胞循环和受损DNA修复中重要的蛋白质相互作用。中心蛋白c端结构域的多个靶点已经被确定，并且该结构域与其中一个靶点结合的结构基础已经被我们的实验室所表征。已经提出了一种模型，其中中心蛋白的c端结构域在钙的基础水平上作为靶蛋白的锚点，n端结构域作为钙信号的传感器。n端结构域的靶标和结合模式仍有待鉴定和表征。我们假设该结构域具有不同于c端结构域的靶特异性和作用方式。我们的研究目标是确定n端结构域的结合伙伴，并使用结构方法表征其与特定靶标相互作用的方式。我们的具体合作目标是：1)确定中心蛋白n端结构域的结合伙伴。酵母-双杂交筛选将用于鉴定潜在的蛋白质相互作用，在这些试验中使用酿酒酵母、莱茵哈地大肠杆菌和智人中心蛋白的结构域和全长构建体作为诱饵。2)测定相互作用蛋白对中心蛋白的亲和力。结合将通过CD或荧光光谱直接监测，并根据这些数据计算中心结构域的相对亲和力。此外，将使用类似的方法评估每种相互作用对钙的依赖性。3)确定中心-靶配合物的结构。我们已经确定了中心蛋白的n端和c端结构域，使用核磁共振波谱获得的数据。我们将利用核磁共振技术来获得中心素与其靶标之间相互作用的特定位点信息。结合蛋白靶的中心蛋白的结构测定将使用x射线晶体学或核磁共振进行。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Centrin is an essential component of the microtubule organizing centers (MTOC), which mediates chromosome segregation during mitosis. In addition to interacting with other MTOC components, it is known to interact with proteins important in both cell cycling and repair of damaged DNA. Multiple targets for the C-terminal domain of centrin have been identified, and the structural basis for binding by this domain to one of its targets has been characterized by our laboratory. A model has been proposed in which the C-terminal domain of centrin serves as an anchor to target proteins at the basal level of calcium and the N-terminal domain serves as the sensor of calcium signals. Targets and the mode of binding for the N-terminal domain remain to be identified and characterized. We hypothesize that this domain has target specificity and mode of action that is distinct from the C-terminal domain. The goal of our research is to identify binding partners for the N-terminal domain, and, using structural methods, to characterize the way in which it interacts with its specific targets. Our specific aims for this collaboration are: 1) Identify binding partners for N-terminal domain of centrin. The Yeast-Two-Hybrid screen would be used to identify potential protein-protein interactions, using domain and full-length constructs for S. cerevisiae, C. rheinhardii and H. sapiens centrins as bait in these assays. 2) Measure the affinity of interacting proteins for centrin. Binding will be monitored directly by CD or fluorescence spectroscopy, and relative affinities for centrin domains calculated from these data. In addition, the calcium dependence of each interaction will be assessed, using similar methods. 3) Determine the structure of centrin-target complexes. We have determined structures of both the N-terminal and C-terminal domains of centrin, using data obtained by NMR spectroscopy. We will make use of NMR techniques to obtain site specific information for interactions between centrin and its targets. Structural determinations of centrin bound to protein targets will be made using X-ray crystallography or NMR.