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: 6541681
• 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/6541681
• 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 structure determination of this protein. 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.

GTP 结合蛋白、其受体及其在质膜上的效应子之间的相互作用已得到很好的表征。相比之下，高尔基膜中类似系统的具体相互作用和功能仍不清楚。选择 G Alpha 相互作用蛋白 (GAIP) 作为模型来研究这种相互作用。 GAIP 与位于高尔基体膜的 Gai3 发生特异性相互作用。构建了含有GAIP核心结构域（150个残基）的质粒构建体。 GAIP 的核心结构域包含在新的 G 蛋白信号传导调节因子（RGS 蛋白）家族中发现的同源结构域。人类 GAIP 的三维折叠已使用 NMR 光谱法测定。 GAIP 结构的细化正在进行中。浓度高于 0.1 mM 的人 GAIP 在溶液中以二聚体形式存在。这导致有效分子量约为 34 kD。确定初始折叠时无需进一步对蛋白质进行氘化，这通常是通过溶液 NMR 来确定这种大小的蛋白质的结构。人类 GAIP 的骨干动态研究也已使用 NMR 进行。这证实了我们的发现，GAIP 在溶液中以二聚体形式存在，至少浓度高于 0.1mM。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。动态数据表明可能存在允许结构构象变化的灵活性。表达 G ai3 亚基的并行项目已经启动。目标是能够在体外重建人类GAIP及其G蛋白补体并观察其生化特性。我们已经完成了人GAIP的溶液结构，并与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 蛋白的催化激活是通过复杂结构的稳定，而不是通过 RGS 与 Galpha 活性位点的直接相互作用。此外，我们还表明，螺旋 V 和 VI 之间接触 Galpha 的环仅在 N 端部分的结构上有所不同。该环的 C 端部分在结合 Galpha 时不会采用不同的构象。我们正在完成这种蛋白质的动态研究。我们还启动了钙结合蛋白 CALNUC 的结构研究。这种蛋白质在钙负载状态下与高尔基体中的 Galpha 结合。据信，CALNUC 通过其与 Galpha 的相互作用来调节高尔基体中的钙浓度。 CANUC 似乎不会影响 Galpha 中的 GTP 水解。因此我们假设有几种不同的与 Galpha 结合的模式。这些不同的模式控制着 Galpha 响应特定刺激而承担的不同功能的子集。我们构建了包含两只 EF 手的 CALNUC 质粒。我们已经成功表达了该蛋白质，并进行了钙结合和肽结合实验。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，蛋白质在两种构象之间经历了一定程度的交换，导致共振信号变宽。然而，我们已经能够确定钙结合和肽结合的特异性。我们目前正在识别同时存在的不同构象。有趣的是，这种交换过程似乎并不影响 CALNUC 结合钙或其靶肽的能力。 我们正在收集 NMR 数据以确定钙存在下人 CALNUC 的溶液结构。我们已经完成了该蛋白的二级结构测定。同时，我们表达 15N 和 13C 标记的 Gai3，以对 Gai3 在分子的各种功能状态下进行结构和动态研究。