Protein Phosphorylation as a Biophysical Switch: NMR Determination of Structural and Dynamic Responses to Phosphorylation

本研究探讨了蛋白质可逆磷酸化调控的生物物理基础。高分辨率多维核磁共振技术将被用来阐明模型系统中蛋白质磷酸化的结构和动力学方面。两个来自鸡c-Src(Src)的蛋白质片段将被研究，它们包含关键的调节域，以研究在该系统中负责控制的生物物理机制。确定第一个片段的结构和动力学，由Src同源3(SH3)和Src同源2(SH2)结构域组成，将起到双重作用：a)它将为建立更多的Src大片段的ADVA NCED研究提供基础；b)它将能够识别分子内相互作用，这可能有助于最近建立的SH3结构域在稳定SH2/磷酸肽相互作用中的作用。第二个更大的片段含有额外的80个残基，组成了Src独特的N-末端区域，将被用来研究蛋白质对磷酸化的结构和动态响应。该片段含有特定的磷酸化位点，已知可以调节Src的酪氨酸激酶活性。这个蛋白质片段在磷酸化和非磷酸化状态下的结构和动力学将使用现在标准的多维技术进行研究。这些研究将揭示被破坏的相互作用和由磷酸化形成的相互作用，并将提供由这一重要的共价修饰在这些常见调控模块中诱导的变化的原子水平的视图。已知%的蛋白质磷酸化参与控制一系列不同的生物过程，包括代谢、细胞增殖和分化、膜运输、基因表达、运动、记忆和学习。然而，通过磷酸化调节的生物物理基础清楚地只对两种蛋白质建立，这两种蛋白质都表现出不同的控制机制。因此，模型体系在磷酸化和非磷酸化状态下的高分辨率结构确定将提供迫切需要的信息，这些信息将有助于理解导致这种普遍存在的蛋白质调控开关的原子水平事件。以Src同源2(SH2)和Src同源3(SH3)结构域为代表的相对较小的保守蛋白结构域在大量信号蛋白中被发现，并被证明是作为模块结合结构域发挥作用的，可以调节分子间和分子内的相互作用。Src蛋白是一种细胞原癌蛋白，也是一种典型的蛋白酪氨酸激酶，它同时含有SH3和SH2结构域以及几个不同的调节磷酸化位点。在Src中，SH2的相互作用被证明是由邻近的SH3结构域以及特定的Ser和远离初级序列的残基的磷酸化所介导的。拟议的研究涉及磷酸化和非磷酸化形式的Src调控装置的结构和动力学研究，并将使用最近开发的高分辨率多维核磁共振波谱技术。总体而言，这些研究将使受磷酸化调控的蛋白质的结构、动力学和功能之间的关系得以建立，并将对许多其他利用磷酸化作为生物物理开关的蛋白质具有广泛的意义。

This research addresses the biophysical basis of protein regulation by reversible phosphorylation. High resolution multidimensional nuclear magnetic resonance (NMR) techniques will be employed to elucidate the structural and dynamic aspects of protein phosphorylation in a model system. Two protein fragments derived from chicken c-Src (Src) that contain key regulatory domains will be studied to investigate the biophysical mechanisms responsible for control in this system. Determination of the structure and dynamics of the first fragment, composed of the Src- homology 3 (SH3) and Src-homology 2 (SH2) domains, will serve a dual purpose: a) it will provide a foundation on which to build more adva nced studies of larger fragments of Src, and b) it will enableidentification of intramolecular interactions that may contribute to the recently established role of the SH3 domain in stabilizing SH2/phosphopeptide interactions. The second and larger fragment, which has an additional 80 residues that comprise the unique N-terminal region of Src, will be utilized to investigate the structural and dynamic response of the protein to phosphorylation. This fragment contains specific phosphorylation sites that are known to regulate the tyrosine kinase activity of Src. The structure and dynamics of this protein fragment in phosphorylated and unphosphorylated states will be pursued using the now-standard multidimensional techniques. These studies will reveal interactions that are disrupted and those that are formed by phosphorylation, and will provide an atomic level view of the changes induced in these common regulatory modules by this important covalent modification. %%% Protein phosphorylation is known to be involved in the control of a diverse array of biological processes, including metabolism, cell proliferation and differentiation, membrane transport, gene expression, locomotion, memory, and learning. However, the biophysical basis of regulation by phosphorylation is clearly established for only two proteins, both of which exhibit different mechanisms of control. Hence, high resolution structure determinations of a model system in phosphorylated and unphosphorylated states will provide much needed information that will aid in understanding of the atomic level events responsible for this ubiquitous protein regulatory switch. Relatively small, conserved protein domains, typified by the Src-homology 2 (SH2) and Src-homology 3 (SH3) domains, are found in a large number of signaling proteins and have been shown to function as modular binding domains that can mediate both inter-and intramolecular interactions. The Src protein, a cellular proto-oncoprotein and a paradigm protein-tyrosine kinase, con tains both SH3 and SH2 domains as well as several different regulatory phosphorylation sites. In Src, SH2 interactions have been shown to be mediated by nearby SH3 domains and by phosphorylation of specific Ser and The residues remotely located in the primary sequence. The proposed research involves structural and dynamics studies of the Src regulatory apparatus in phosphorylated and unphosphorylated forms, and will employ recently developed high resolution multidimensional NMR spectroscopic techniques. Overall, these studies will enable relationships between structure, dynamics and function in a protein regulated by phosphoylation to be established, and will have broad implications for numerous other proteins that utilize phosphorylation as a biophysical switch.