CRYSTAL STRUCTURE OF THE ENTIRE EXTRACELLULAR DOMAIN OF C-KIT RECEPTOR

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。受体酪氨酸激酶（RTKs）的细胞信号传导在细胞周期、细胞迁移、细胞代谢、细胞存活、细胞增殖和细胞分化等许多细胞过程的控制中起着关键作用。c-Kit受体是一种RTK，在许多组织中表达，并通过与其配体干细胞因子（SCF）的相互作用介导其生物效应。SCF结合导致c-Kit二聚化，刺激自磷酸化和激活内在蛋白激酶活性。尽管对c-Kit信号传导的分析已经做了很多工作，但在缺乏SCF的情况下，受体如何避免二聚化和激活的重要机制问题仍未得到解答。为了进一步了解c-Kit的自抑制和激活机制，我们提出解决c-Kit的胞外结构域在其未占据形式的结构。与之前的方案相比，下面的表达和纯化程序有了很大的改进。利用波浪生物反应器在Sf9昆虫细胞中表达了包含5个ig结构域的c-Kit的整个胞外结构域。15株凋落物经金属螯合层析后的表达量为30 mg粗糖基化蛋白。在使用内糖苷酶F1酶和阴离子交换色谱进行去糖基化处理后，浓缩15mg均质（通过SDS和Native-PAGE估计）去糖基化蛋白并冷冻。经过广泛筛选（每种形式600个条件），发现了未占用的去糖基化c-Kit受体的结晶条件。未占用c-Kit的初始结晶条件为10%聚乙二醇1000，pH 6.0。优化结晶条件后，晶粒形状和尺寸均得到改善，晶粒尺寸在75 ~ 150 μ m之间。使用家用衍射仪（Rigaku R-Axis 4），曝光90分钟，晶体衍射高达8 A，斑点可以检测到高达5 A。最近我们收集了NSLS x29波束线的数据。我们得到了几个原生数据集，达到3.0 A，完整性达到96%。晶体属于菱形空间群R3，单位胞维a = b = 162, c = 66 a。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell signaling by receptor tyrosine kinases (RTKs) plays a pivotal role in the control of many cellular processes including the cell cycle, cell migration, cell metabolism, cell survival, cell proliferation and cell differentiation. The c-Kit receptor is an RTK that is expressed in many tissues and mediates its biological effects following interactions with its ligand, stem cell factor (SCF). SCF binding leads to c-Kit dimerization, stimulation of autophosphorylation and activation of the intrinsic protein kinase activity. Although much work has been devoted to analysis of c-Kit signaling, important questions of mechanism regarding how the receptor avoids dimerization and activation in the absence of the SCF, remain unanswered. In order to learn more about the auto inhibition and activation mechanisms, we propose to solve the structure of the extracellular domain of c-Kit in its unoccupied form. The following expression and purification procedure is considerably improved in comparison to previous protocol. The entire extracellular domain of c-Kit containing five Ig-domains is expressed in Sf9 insect cells using wave bioreactor. The expression yield from 15 litter culture is 30 mg of crude glycosylated protein after metal-chelating chromatography. Following deglycosylation treatment using Endoglycosydase F1 enzyme and anion exchange chromatography, 15 mg of a homogeneous (estimated by SDS and Native-PAGE) deglycasylated protein is concentrated and frozen. After extensive screening (600 conditions for each form), crystallization conditions were found for unoccupied deglycosylated c-Kit receptor. The initial crystallization conditions for the unoccupied c-Kit are 10% polyethyleneglycol 1000 at pH 6.0. After its crystallization conditions were optimized, crystal shapes and sizes are improved with size varies from 75-150 microns. Using home-source diffractometer (Rigaku R-Axis 4), with 90 minutes exposure, the crystals diffract up to 8 A and spots can be detected upto 5 A. Recently we collected data on the NSLS x29 beam line. We got couple of native data sets upto 3.0 A with 96% completeness. The crystals belong to the rhombohedral space group R3, with unit-cell dimensions a = b = 162, c = 66 A.