DOMAIN SPECIFIC INTERACTIONS OF CENTRIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Centrin是微管组织中心(MTOC)的重要组成部分，在有丝分裂过程中调节染色体分离。除了与其他MTOC组分相互作用外，已知它还与细胞周期和受损DNA修复中重要的蛋白质相互作用。中心素C-末端结构域的多个靶点已经被确定，我们的实验室已经表征了该结构域与其一个靶点结合的结构基础。已经提出了一个模型，在该模型中，Centin的C-末端结构域作为钙基础水平的靶蛋白的锚定，N-末端结构域作为钙信号的传感器。N-末端结构域的靶点和结合方式仍有待确定和表征。我们假设该结构域具有不同于C-末端结构域的靶向性和作用模式。我们研究的目标是确定N-末端结构域的结合伙伴，并使用结构方法来表征它与其特定靶点相互作用的方式。我们这次合作的具体目标是：1)寻找Centin N-末端结构域的结合伙伴。酵母双杂交筛选将被用来确定潜在的蛋白质-蛋白质相互作用，在这些分析中使用酿酒酵母、类风湿杆菌和智人中心蛋白的结构域和全长结构作为诱饵。2)测定相互作用蛋白对中心素的亲和力。结合将直接通过CD或荧光光谱进行监测，并根据这些数据计算中心素域的相对亲和力。此外，还将使用类似的方法评估每个相互作用对钙的依赖程度。3)确定了中心-靶复合体的结构。我们利用核磁共振波谱数据确定了Centin的N末端和C末端结构域的结构。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。将使用X射线结晶学或核磁共振来确定与蛋白质靶标结合的中心素的结构。

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.