Nmr Studies Of The Regulation Of Cell Signaling

细胞信号传导调节的核磁共振研究

• 批准号: 6690467
• 负责人: NICO TJANDRA
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6690467
• 关键词: G protein; Golgi apparatus; biological signal transduction; calcium binding protein; conformation; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatase activating protein; nuclear magnetic resonance spectroscopy; protein folding; protein protein interaction; protein structure function; structural biology

The interaction between the GTP-binding protein, its receptor, and its effector at the plasma membrane is well characterized. In contrast, the specific interaction and function of similar systems in the Golgi membranes is still not clear. A G Alpha Interacting Protein (GAIP) was chosen as a model to study this interaction. GAIP interacts specifically with the Gai3 which has been localized to the Golgi membranes. A plasmid construct containing the core domain (150 residues) of GAIP was constructed. The core domain of GAIP contains a homology domain found in a novel family of regulators of G protein signaling (RGS proteins). The three dimensional fold of human GAIP has been determined using NMR spectroscopy. The refinement of the structure of GAIP is in progress. Human GAIP at a concentration higher than 0.1 mM exists as a dimer in solution. This results in an effective MW of roughly 34 kD. The initial fold was determined without further deuteration of the protein which is typically done for structure determination of proteins of this size by solution NMR. A backbone dynamic study of human GAIP has also been carried out using NMR. This confirms our finding that GAIP exists as a dimer in solution, at least at concentrations higher than 0.1mM. The dynamic data also reveals the regions which have flexibility. Initial comparison of GAIP and the X-ray structure of RGS4 complexed to Gai1 reveals some conformational changes upon binding to the G protein. The dynamic data suggests possible flexibility that allows the conformational change in the structure. A parallel project to express the G ai3 subunit has been initiated. The goal is to be able to reconstruct human GAIP and its G protein complement in vitro and observe the biochemical properties. We have completed the solution structure of human GAIP and carried out detailed comparison to the structure of RGS4 complexed to Galpha-i. We concluded that the activation of catalysis by RGS protein is through stabilization of the complex structure, not by direct interaction of RGS to the active site of Galpha. Furthermore, we have shown that the loop between helix V and VI which contacts the Galpha differs in structure only for the N-terminal portion. The C-terminal portion of this loop does not adopt a different conformation upon binding the Galpha. We are finishing the dynamic study of this protein. We have also initiated a structural study of a calcium binding protein, CALNUC. This protein in the calcium loaded state binds Galpha in the Golgi. It is believed that CALNUC is regulated through its interaction with Galpha to modulate calcium concentration in the Golgi apparatus. CALNUC does not seem to effect the GTP hydrolysis in Galpha. Therefore we hypothesize that there are several different modes of binding to the Galpha. These different modes govern a subset of different functions that the Galpha would undertake to respond to a certain stimulus. We have constructed the CALNUC plasmid which encompasses the two EF hands. We have succesfully expressed the protein and have carried out experiments on calcium binding as well as peptide binding. The peptide used represents the C-terminal helix of the Gai. Our results so far show that the protein undergoes a certain degree of exchange between two conformations that results in broadening of the resonance signals. However, we have been able to establish the specificity of calcium binding as well as peptide binding. We are currently identifying the different conformations that simultaneously exist. It is interesting that this exchange process does not seem to effect CALNUC's ability to bind calcium or its target peptide. We are collecting NMR data to determine the solution structure of human CALNUC in teh presence of calcium. We have finished the secondary structrue determination of this protein. We also have completed collection of data for the determination of the three dimensional structure of CALNUC. At the same time we are expressing 15N and 13C labeled Gai3 to carry out structural as well as dynamic studies of the Gai3 in the various functional states of the molecule. So far we have been able to express the protein and currently are working on a purification protocol to provide suitable sample for study under NMR condition. In addition we have started the expression of AGS3. It is a protein that contains several goLoco domains. Ags3 seems to have an opposite function than RGS domain, that is it slows the turnover of the Gai into its inactive state. The study of this protein will provide a wider picture of all possible binding modes of the Ga in its function to response to various cell signals.

GTP结合蛋白，其受体和其在质膜处的效应子之间的相互作用得到了很好的特征。相反，高尔基膜中相似系统的特定相互作用和功能仍然不清楚。选择Gα相互作用蛋白（GAIP）作为研究这种相互作用的模型。 GAIP与已定位在高尔基膜的GAI3专门相互作用。构建了含有GAIP的核心结构域（150个残基）的质粒构建体。 GAIP的核心结构域包含一个在G蛋白信号传导（RGS蛋白）的新型调节剂家族中发现的同源域。使用NMR光谱法确定了人GAIP的三维折叠。 GAIP结构的完善正在进行中。作为溶液中的二聚体存在于高于0.1 mm的浓度下的人GAIP。这会导致大约34 kD的有效MW。确定初始折叠，而无需进一步的蛋白质呈现，该蛋白通常用于通过溶液NMR来确定此大小的蛋白质的结构。还使用NMR进行了对人GAIP的主干动态研究。这证实了我们的发现，即GAIP作为溶液中的二聚体存在，至少在高于0.1mm的浓度下存在。动态数据还揭示了具有灵活性的区域。 GAIP和与GAI1复合的RGS4的X射线结构的初步比较揭示了与G蛋白结合后的一些构象变化。动态数据表明可能允许结构构象变化的灵活性。已经启动了表达G AI3亚基的平行项目。目的是能够在体外重建人GAIP及其G蛋白补体，并观察生化特性。我们已经完成了人GAIP的溶液结构，并与复合到Galpha-I复合的RGS4的结构进行了详细的比较。我们得出的结论是，RGS蛋白催化的激活是通过稳定复合结构的稳定，而不是通过RGS与Galpha活性位点的直接相互作用。此外，我们已经表明，接触Galpha的螺旋V和VI之间的循环仅在N末端部分的结构上有所不同。该回路的C末端部分在结合Galpha时不会采用不同的构象。我们正在完成该蛋白质的动态研究。我们还启动了钙结合蛋白Calnuc的结构研究。该蛋白在钙加载状态下结合高尔基体中的galpha。据信，CALNUC通过与Galpha的相互作用来调节Calnuc以调节高尔基体中的钙浓度。 CALNUC似乎不会影响Galpha中的GTP水解。因此，我们假设与Galpha有几种不同的结合模式。 These different modes govern a subset of different functions that the Galpha would undertake to respond to a certain stimulus.我们已经构建了包含两个EF手的Calnuc质粒。我们已经成功地表达了蛋白质，并在钙结合以及肽结合上进行了实验。所使用的肽代表GAI的C末端螺旋。到目前为止，我们的结果表明，蛋白质在两个构象之间经历了一定程度的交换，从而扩大了共振信号。但是，我们已经能够建立钙结合和肽结合的特异性。我们目前正在确定同时存在的不同构象。有趣的是，这种交换过程似乎没有影响Calnuc结合钙或其靶肽的能力。我们正在收集NMR数据，以确定钙的存在中人Calnuc的溶液结构。我们已经完成了该蛋白质的二次结构测定。我们还完成了数据收集，以确定CALNUC的三维结构。同时，我们表达了15N和13C标记的GAI3，以在分子的各个功能状态下对GAI3进行结构和动态研究。到目前为止，我们已经能够表达蛋白质，目前正在制定纯化方案，为NMR条件下的研究提供合适的样本。此外，我们已经开始了AGS3的表达。它是一种蛋白质，其中包含多个戈洛科结构域。 AGS3似乎比RGS域具有相反的功能，也就是说，它将GAI的营业额放慢了其非活性状态。对该蛋白质的研究将为GA的所有可能结合模式提供对各种细胞信号的响应的更广泛图像。