Antizyme and ornithine decarboxylase: Interactions and crystallization

本项目旨在研究鸟氨酸脱羧酶（ODC）的独特调控，ODC是多胺生物合成的关键限速酶，涉及一种独特的蛋白质，称为抗酶。Antizyme与ODC结合，抑制ODC活性，并促进ODC被26 S蛋白酶体降解，而不发生多聚泛素化。酿酒酵母抗酶（AZ）与哺乳动物AZ相似之处在于其翻译移码合成模式以及其抑制和促进鸟氨酸脱羧酶（ODC）降解的能力。尽管移码位点存在保守结构域，但酵母和哺乳动物AZ的氨基酸序列差异很大。尽管对AZ和ODC的相互作用进行了许多研究，但ODC：AZ复合物尚未从任何来源纯化，因此仍然缺乏关于复合物化学计量的明确信息以及结构相互作用的细节。在我们的初步研究中，我们已经报道了蛋白质的生物化学和生物物理特性，以及由此产生的ODC：AZ复合物。全长抗酶的远紫外CD光谱显示酵母蛋白由51%的β-折叠、19%的α-螺旋和24%的卷曲组成。表面等离子体共振分析表明，酵母AZ与酵母ODC的结合常数为6 × 107（M ~（-1））。使用纯化的His标记的AZ作为结合伴侣，我们纯化了ODC：AZ抑制复合物。分离的复合物没有ODC活性。该复合物的分子量为90 kDa，这表明AZ和ODC在体外以一比一的化学计量结合。两个单独的蛋白质和ODC：AZ复合物的圆二色性（CD）光谱的比较显示复合物中二级结构的变化。 目前，我们与大卫戴维斯博士的实验室和Udesh de Silva博士（分子生物学实验室）合作，试图使AZ：ODC复合物结晶。对于这项研究，我们已经开发了一种方法，快速共纯化AZ：ODC复合物从粗提物，而无需单独纯化两种蛋白质。我们还建立了一个菌株，在同一E.因此，当我们在体内诱导AZ和ODC蛋白在同一细胞中共表达并结合在一起时。使用该制备物，我们在抗酶和抗酶-ODC复合物的晶体筛选中获得了几个命中。 在一个相关的项目中，我们正在通过氘交换详细研究酵母抗酶和鸟氨酸脱羧酶的蛋白质-蛋白质相互作用。为此，我们从大肠杆菌中纯化了酵母抗酶、鸟氨酸脱羧酶和抗酶-ODC复合物。大肠杆菌表达系统中进行氘交换和质谱分析，以找出抗酶和ODC中相互作用的残基。对于本研究，我们已将蛋白质样本发送至NIC-Frederick LPAT-质谱机构（Timothy Veenstra博士和Ming Zhou博士）。在D2 O缓冲液中进行H/D交换实验，并通过质谱法分析肽。从这些分析的初步数据显示，许多相互作用的残基从两种蛋白质的抗酶-ODC复合物。现在正在重复这些研究，并且基于这些研究，将通过定点诱变产生突变体，以进一步确认每个分子中的相互作用残基，并创建分子模型以显示酵母抗酶和ODC相互作用的细节。

This project aims to investigate the unique regulation of ornithine decarboxylase (ODC), the key rate-limiting enzyme of polyamine biosynthesis, involving a unique protein termed antizyme. Antizyme binds to ODC, inhibits ODC activity and facilitates the degradation of ODC by the 26S proteasome without polyubiquitination. Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite the presence of conserved domains in the frameshifting site, the amino acid sequences of yeast and mammalian AZ differ considerably. Despite many studies on the interaction of AZ and ODC, the ODC: AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex were still lacking as well as details in the structural interactions. In our initial study, we have reported the biochemical and biophysical characteristics of the proteins, and the resulting ODC: AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% beta-sheet, 19% alpha-helix, and 24% coils. Surface plasmon resonance analyses show that theassociation constant (KA) between yeast AZ and yeast ODC is 6x 107 (M-1). Using purified His-tagged AZ as a binding partner, we have purified the ODC: AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC: AZ complex shows a change in the secondary structure in the complex. Currently, in collaboration with Dr. David Davies' laboratory, and Dr. Udesh de Silva (Laboratory of Molecular Biology) we are trying to crystallize the AZ:ODC complex. For this study, we have developed a method to rapidly co-purify the AZ:ODC complex from crude extracts without purifying the two proteins separately. We have also developed a strain to overexpress ODC and antizyme in the same E. coli expression system so that when we induce the proteins both AZ and ODC co-express and bind together in the same cell in vivo. Using this preparation we obtained several hits in our crystal screens for both antizyme and antizyme-ODC complex. In a related project, we are studying in detail the protein-protein interaction of yeast antizyme and ornithine decarboxylase by deuterium exchange. For this we have purified yeast antizyme, ornithine decarboxylase and antizyme-ODC complex from the E. coli expression systems and analyze the interaction by deuterium exchange followed by mass spectrometric analyses to find out the interacting residues in antizyme and ODC. For this study we have sent protein samples to the NIC-Frederick LPAT-Mass Spectroscopy facility (Dr. Timothy Veenstra and Dr. Ming Zhou). H/D exchange experiments have been performed in D2O buffer and the peptides were analyzed by mass spectrometry. Preliminary data from these analyses show many interacting residues from both the proteins in the antizyme-ODC complex. These studies are now being repeated and based on these studies mutants will be generated by site directed mutagenesis to further confirm the interacting residues in each molecule and to create a molecular model to show details of the yeast antizyme and ODC interaction.