Role of three dimensional structure of proteins in signal transduction

本研究的目的是从结构上阐明蛋白质在信号转导过程中的作用。我们还开发了蛋白质表达和纯化系统，以制备结构研究所需的大量蛋白质。在本研究项目的过程中，通过核磁共振光谱或X射线结晶学确定了许多蛋白质的结构。分别测定了SOS富含脯氨酸多肽和Shc富含磷酸酪氨酸多肽与Grb2n-SH3和SH2结构域的络合作用，阐明了识别靶序列的一般机制。用核磁共振法测定了其三维结构。虽然destrin家族蛋白和明胶蛋白家族蛋白之间的序列不存在同源性，但两者的三维结构非常相似，这表明肌动蛋白的切断机制是相同的。由此，提出了肌动蛋白纤维断裂的一般机制。核磁共振测定了中期因子的三维结构，阐明了肝素低聚糖形成二聚体的机理。中期因子二聚体的形成似乎对受体的激活及其生物学功能是必不可少的。我们还确定了PKC的CRD结构域，并提出了识别DAG的机制。通过X射线结晶学分析，确定了细菌信号转导系统的一些蛋白质结构，包括信号转导系统的发射域和接收域。我们还建立了N-WASP、Tob和CAF1的蛋白表达和纯化系统。N-WASP是对生长因子受体信号的响应而形成的声带的关键。TOB和CAF1在细胞周期中起着关键作用。对这两种蛋白质的结构研究都开始了。

The aim of the present research was to elucidate the roles of proteins in signal transduction process on the structural basis. We also developed the protein expression and purification systems for preparation of amount of proteins required for structural studies. In the course of the present research project, a number of protein structures were determined by either NMR spectroscopy or X-ray crystallography. Grb2 n-SH3 and SH2 domains complexed with proline rich peptide from Sos and phosphotyrosine containing peptide from Shc, respectively were determined and the general mechanism for recognition of target sequences were elucidated. Three dimensional structure of destrin was determined by NMR. Although the sequence homology between destrin family and gelsolin family proteins was not present, the three dimensional structure of both proteins were very similar, suggesting the same mechanism for actin severing. Thus, the general mechanism for severing of the actin fiber was proposed. The three dimensional structure of midkine was also determined by NMR and elucidated the mechanism of dimer formation on heparin oligosaccharides. The dimer formation of midkine seems to be essential for activation of the receptor and its biological function. We also determined the CRD domain of PKC and suggested the mechanism of DAG recognition. By X-ray crystallography, a number of protein structures were determined including transmitter and receiver domains of bacterial signal transduction system. We also made protein expression and purification system for N-WASP, Tob and CAF1. N-WASP is essential for formation of philopodia in response to the growth factor receptor signal. Tob and CAF1 play pivotal role in cell cycling. The structural studies started for both proteins.