Mapping the Binding Surfaces of Ras and Related GTPases

描述(由申请人提供)：RAS是一种参与控制细胞增殖的小GTP酶，也是本研究提案的重点。它的几个突变与人类癌症直接相关，因为它们仍然处于激活状态，导致不受控制的细胞分裂。RAS是一组结构相似的蛋白质中的一员，参与与调节剂的高度特异性相互作用，这些调节剂在复杂的信号转导级联中控制它们与靶蛋白的结合能力。我们的目标是破译RAS家族中GTP酶的分子识别规则。这些规则将在未来用于指导针对有缺陷的致癌RAS蛋白的特定配体的设计。我们的方法是基于对RAS的结合表面的比较分析，RAS是它最常见的两个致癌突变体，以及它的近亲家族成员Rap和ral。传统的X射线结晶学将与多溶剂晶体结构(MSCS)方法相结合，其中有机溶剂分子被用作蛋白质结合位点的分子表面特征的探针。使用这些方法，我们计划创建RAS、RasG12V、RasQ61L、Rap和Ral结合表面的“功能图”。我们将通过四个具体目标实现我们的目标。首先，我们将使用MSCS方法来鉴定RAS、RAP和RAL上的主要靶标识别位点和可能的二级结合位点，以便将整个家族的共同结合位点特征与每个家族成员特有的结合位点特征区分开来。其次，我们将使用MSCs方法来确定由于致癌突变G12V和Q61L而导致的RAS结合位点的关键差异。第三，我们将解决Ral及其与其靶标Ral结合蛋白1(RalBP1)的Ral最小结合结构域的络合物的晶体结构。我们将把这种结构与先前发表的RAS(RAS/RalGDS)和Rap(Rap/Raf)的类似络合物进行比较。第四，我们将结合前三个目标的结果来描述RAS的特定区域，特别是其致癌突变。其目标是为基于提供多角度的多结构的配体设计和发现提供关键信息。

DESCRIPTION (provided by applicant): Ras is a small GTPase involved in the control of cell proliferation and is the focus of this research proposal. Several of its mutants have been directly linked to human cancers, as they remain in an activated state that leads to uncontrolled cell division. Ras is a member of a group of structurally similar proteins involved in highly specific interactions with regulatory agents that control their ability to bind to target proteins within complicated signal transduction cascades. Our objective is to decipher the rules for molecular recognition within the Ras family of GTPases. These rules will be used in the future to guide the design of specific ligands targeted towards the defective oncogenic Ras proteins. Our approach is based on a comparative analysis of the binding surfaces of Ras, two of its most common oncogenic mutants and of its close family members Rap and Ral. Conventional X-ray crystallography will be combined with the multiple solvent crystal structures (MSCS) method where organic solvent molecules are used as probes to the molecular surface features of protein binding sites. Using these methods, we plan to create "functionality maps" of the binding surfaces of Ras, RasG12V, RasQ61L, Rap and Ral. We will accomplish our goals through four specific aims. First, we will use the MSCS method to characterize both the primary target recognition sites and the putative secondary binding sites on Ras, Rap and Ral, so that the common binding site features across the family can be distinguished from those that are specific to each family member. Second, we will use the MSCS method to determine key differences in the binding site of Ras due to the oncogenic mutations G12V and Q61L. Third, we will solve the crystal structure of Ral and of its complex with the Ral minimum binding domain of its target Ral Binding Protein 1 (RalBP1). We will compare this structure with the analogous complexes for Ras (Ras/RalGDS) and Rap (Rap/Raf), which have previously been published. Fourth, we will combine the results from the first three aims to delineate areas specific to the Ras, and, in particular, to its oncogenic mutants. The goal is to provide key information for ligand design and discovery that is based on multiple structures that offer multiple perspectives.