Structure/function Study Of Viaf

Viaf的结构/功能研究

• 批准号: 6501188
• 负责人: DENNIS A TORCHIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 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/6501188
• 关键词: apoptosis; cell growth regulation; nuclear magnetic resonance spectroscopy; protein structure function; proteins; tissue /cell culture

VIAF is a member of a conserved protein family (initially identified by our collaborators in Colin Duckett's lab in NCI), that associate with animal IAPs (inhibitor of apoptosis proteins). VIAF itself substantially protects cells from Fas- and Bax-induced apoptosis, while coexpression of VIAF with suboptimal quantities of XIAP confers almost complete protection from these inducers. VIAF and XIAP activate JNK in a synergistic manner. Hence, VIAF is a novel cofactor which modulates the anti-apoptotic and signaling properties of the IAP family. In order to obtain a basis for understanding the function of VIAF at the molecular level, we have initiated a determination of the three-dimensional structure of VIAF using NMR spectroscopy. Full length VIAF contains 239 residues, but two-hybrid-screening studies have shown that the C-terminal 128 residue region of the protein is necessary and sufficient for interaction with Op-IAP. Hence we have expressed the C-terminal domain of VIAF, henceforth referred to as VIAFC in E..coli using the pET11 vector. Cultures were grown in minimal media containing either 15N or 15N plus 13C labeled ammonium chloride and glucose, respectively. The protein was purified from inclusion bodies and successfully refolded. NMR spectra were recorded on the protein dissolved in a 10 mM phosphate buffer, pH 7.4, at 35 ?C. The NMR resonances of backbone heavy atoms were assigned using the usual suite of 3D-heteronuclear experiments. Specifically, CBCA(CO)NH, HNCA and HNCACB were used to assign the amide-N, -HN, alpha-C? and beta-C signals. Next, the carbonyl carbons were assigned using the 3D-HNCO experiment. The HCACO experiment was then used to confirm these assignments. The alpha-H? and beta-H signals were then assigned using HNHA and HBHA(CO)NH experiments. These signal assignments were used to predict the secondary structure of VIAF using the chemical shift index (CSI). Surprisingly the VIAF secondary structure was highly homologous to that of phosducin, in spite of the limited sequence homology shared by the two proteins. The predicted secondary structure of VIAFC and the existing crystal structure of phosducin were then used to build an initial model for VIAFC. We next weakly oriented VIAFC in a Pf1 phage liquid crystal medium. Using a 15N-13C double-labeled sample we measured five sets of different heteronuclear residual dipolar couplings. Homology searches using these dipolar couplings were conducted against seven residue fragments generated from a PDB structure file and the best fits for these dipolar couplings yielded a model of the protein structure that had the correct secondary structure. However, additional data, in the form of NOE distance restraints are needed to determine the three dimensional structure of VIAFC. In order to obtain the needed NOE restraints, proton resonance assignments have been extended to the amino acid sidechains using a combination of C(CO)NH, HC(CO)NH, and 3D-HCCH-TOCSY experiments. Currently we have assigned more than 90 % of the protons in the structured region of the protein. We have also acquired 15N and 13C three- and four-dimensional NOESY spectra and are currently analyzing these data sets to obtain proton-proton distance restraints. We have been encouraged by the fact that the VIAFC model based upon the phosducin structure is consistent with the long range NOES we have observed so far. Upon solving the three dimensional solution structure of VIAFC we plan to study the interaction of VIAFC with XIAP. Specifically, we will map out the surface of VIAFC that interacts with XIAP.

Viaf是保守蛋白质家族的成员（最初由我们在NCI的Colin Duckett实验室中的合作者确定），该成员与动物IAP（凋亡蛋白抑制剂）相关。 VIAF本身实质上保护细胞免受FAS和BAX诱导的细胞凋亡的影响，而ViaF与次优量的XIAP共表达几乎完全保护了这些诱导剂。 VIAF和XIAP以协同的方式激活JNK。因此，VIAF是一种新型的辅助因子，可调节IAP家族的抗凋亡和信号传导特性。为了获得理解VIAF在分子水平上的功能的基础，我们使用NMR光谱启动了VIAF的三维结构的确定。 全长VIAF包含239个残基，但两次杂交研究表明，蛋白质的C末端128残基区域是必需的，足以与OP-IAP相互作用。因此，我们表达了VIAF的C末端结构域，因此使用PET11载体在E..Coli中称为Viafc。培养物分别在含有15N或15N加13C标记的最小培养基中生长，分别标记为氯化铵和葡萄糖。该蛋白质从包容体中纯化，并成功地重新折叠。 NMR光谱记录在溶解在10 mm磷酸盐缓冲液中的蛋白质上，pH 7.4，35？c。 使用通常的3D核核实验套件分配了主链重原子的NMR共振。具体而言，使用CBCA（CO）NH，HNCA和HNCACB分配酰胺-N，-HH，Alpha-C？和beta-c信号。接下来，使用3D-HNCO实验分配了羰基碳。然后使用HCACO实验确认这些作业。 alpha-h？然后使用HNHA和HBHA（CO）NH实验分配β-H信号。 这些信号分配用于使用化学移位指数（CSI）预测VIAF的二级结构。令人惊讶的是，尽管两种蛋白质共享有限的序列同源性，但VIAF二级结构与硫糖素的二级结构高度同源。然后使用VIAFC的预测二级结构和现有的硫素晶体结构来构建ViaFC的初始模型。我们接下来在PF1噬菌体液晶培养基中弱定向的ViaFC。使用15N-13C双标签样品，我们测量了五组不同的异核残留偶极耦合。使用这些偶极耦合进行了同源搜索，对从PDB结构文件产生的七个残基片段进行，这些偶极耦合的最佳拟合产生了具有正确二级结构的蛋白质结构的模型。但是，需要以NOE距离限制形式的其他数据来确定VIAFC的三维结构。为了获得所需的NOE约束，使用C（CO）NH，HC（CO）NH和3D-HCCH-TOBOCSE实验的C（CO）NH，HC（CO）组合将质子共振分配扩展到氨基酸SIDECHAINS。目前，我们在蛋白质结构化区域中分配了超过90％的质子。我们还获得了15N和13C的三维噪声光谱，目前正在分析这些数据集以获得质子 - 普罗顿距离限制。我们对基于磷素结构的VIAFC模型与到目前为止观察到的远距离Noes一致的事实感到鼓舞。解决VIAFC的三维溶液结构后，我们计划研究VIAFC与XIAP的相互作用。具体而言，我们将绘制与XIAP相互作用的VIAFC的表面。