STRUCTURAL DETERMINATION OF SHP 1 & PEPTIDE COMPLEXES: IMMUNOLOGY

在过去的一年里，我们进行了晶体研究 由Gia1的催化中心支撑的催化中间体。 与GDP挂钩的复合体的结构已经在一系列问题上得到了确定 在硫酸盐存在下的镁离子浓度，类似于 无机磷酸盐是GTP水解的产物。这些水晶 结构，用从国际象棋A1光束线获得的数据确定， 证明Switch II螺旋的重组是伴随而来的 GTPgS、GDP和GDPPI的b-g磷酸键断裂 使用A1和F1光束线的复合体，揭示了 蛋白质处于所有三种稳定的催化中间状态。这个 RGS4(G蛋白信号转导调节因子)与 Gdp-氟化铝-镁离子结合形式的GIA1也在 2.8分辨率使用来自F2线的辐射。这表示 G蛋白的第一结构--G蛋白GAP(GTP酶激活 蛋白质)复合体，并部分演示了RGS4是如何 稳定GTP水解的过渡态。最近， 刺激性G蛋白α亚基GSA的结构及其功能 与其效应物腺苷酸的可溶性催化结构域形成的络合物 环化酶，已被测定。这些结构，在2.8确定- 2.3(所研究的一系列络合物的)分辨率揭示了 异源三聚体G蛋白与其相互作用方式的研究 效应器，以及三磷酸腺苷在催化部位的结合部位 腺苷环化酶和二萜类激活剂Forsklin的作用。 这些研究利用了在国际象棋A1光束线上测量的数据， 代表了G蛋白结构研究的一个里程碑，并建议 第一次，异三聚体G蛋白是如何激活它们的 效应器。同步加速器辐射是所有 在上述项目中，由于衍射力较弱和 在每个实验中使用的晶体的限制尺寸

Over the past year, we have carried out crystallographic studies of catalytic intermediates supported by the catalytic site of Gia1. The structure of the GDP-bound complex has been determied at a series of Mg2+ concentrations in the presence of sulfate, an analog of inorganic phospate, a product of GTP hydrolysis. These crystal structures, determined with data obtained from the CHESS A1 beam line, demonstrate that restructuring of the switch II helix are concomitant with cleavage of the b-g phosphate bond of GTPgS, GDP and GDPPi complexes using the A1 and F1 beamlines, reveaing the conformation of the protein in all three stable catalytic intermediate states. The complex between RGS4 (Regulator of G protein Signalling) and the GDP-Aluminum fluroride-Mg2+ bound form of Gia1, was also determined at 2.8
resolution using radiation from the F2 line. This represents the first structure of a G protein-G protein GAP (GTPase Activating Protein) complex to be determined, and demonstrates, in part, how RGS4 stabilizes the transition state for GTP hydrolysis. Most recently, the structures of the stimulatory G protein alpha subunit Gsa and its complex with the soluble catalytic domains of its effector, adenylyl cyclase, have been determined. These structures, determined at 2.8 - 2.3
resolutions (for the series of complexes studied) have revealed the mode of interacion between a heterotrimeric G protein and its effector, as well as the binding sites of ATP in the catalytic site of adenylyl cyclase and that of the diterpine activator, forskolin. These studies, enabled with data measured at the CHESS A1 beamlines, represent a milestone in structural studies of G proteins, and suggest for the first time, how heterotrimeric G proteins activate their effectors. Synchrotron radiation was essential to the success of all of the projects described above, due to the weak diffracing power and limiting size of the crystals utilized in each of the experiments