NMR Studies of the Regulation of Cell Signaling

GTP结合蛋白、其受体和其在质膜上的效应器之间的相互作用得到了很好的表征。相比之下，高尔基体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)作为研究这种相互作用的模型。GAIP与定位于高尔基体膜的GAI3发生特异性的相互作用。构建了含有GAIP核心区(150个残基)的表达载体。GAIP的核心区含有一个新的G蛋白信号调节蛋白家族(RGS蛋白)中的同源结构域。用核磁共振波谱测定了人GAIP的三维折叠。全球公认会计准则结构的改进正在进行中。浓度大于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34kD。通过溶液核磁共振测定这种大小的蛋白质的结构时，不需要对蛋白质进行进一步的氢化，就可以确定初始折叠。利用核磁共振对人GAIP的骨架动力学进行了研究。这证实了我们的发现，GAIP在溶液中以二聚体的形式存在，至少在高于0.1 mM的浓度下存在。动态数据还揭示了具有灵活性的区域。初步比较GAIP和与GAI1结合的RGS4的X射线结构，发现与G蛋白结合后发生了一些构象变化。动态数据表明了允许结构构象变化的可能的灵活性。一个表达GAI3亚单位的平行项目已经启动。目的是能够在体外构建人GAIP及其G蛋白补体，并观察其生化性质。我们已经完成了人GAIP的溶液结构，并与与Galpha-I络合的RGS4的结构进行了详细的比较。我们的结论是，RGS蛋白的催化活性是通过稳定复杂的结构来实现的，而不是通过RGS与Galpha活性部位的直接相互作用来实现的。此外，我们已经证明，螺旋V和VI之间与Galpha接触的环在结构上只在N末端部分不同。当与Galpha结合时，该环的C-末端部分不采用不同的构象。我们正在完成对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。这种处于钙负荷状态的蛋白质与高尔基体中的Galpha结合。据认为，CALNUC是通过与Galpha的相互作用来调节高尔基体中的钙浓度的。CALNUC似乎不影响GTP在Galpha中的水解。因此，我们假设存在几种不同的与Galpha结合的模式。这些不同的模式管理着Galpha将承担的对特定刺激做出反应的不同功能的子集。我们已经构建了包含两个EF手的CALNUC质粒。我们成功地表达了该蛋白，并进行了钙结合和多肽结合的实验。所用的肽代表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，导致了共振信号的展宽。然而，我们已经能够确定钙结合和多肽结合的特异性。我们目前正在鉴定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。

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 Vand 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 thatthere 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 successfully 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.