Antizyme and ornithine decarboxylase: Interactions and crystallization

鸟氨酸脱羧酶和抗酶相互作用界面显示了对鸟氨酸脱羧酶活性抑制和结合重要的关键残基 鸟苷酸脱羧酶（ODC）是多胺生物合成的关键限速酶。多胺是阳离子分子，在细胞增殖、蛋白质合成和基因调控中起重要作用，并且在从细菌到人类的生物体中发现。在各种生物体中，ODC活性由其抑制性蛋白抗酶（AZ）进行后调节，所述抑制性蛋白抗酶（AZ）是由于多胺浓度增加而引起的核糖体移码的结果而合成的。AZ表达为单体，其通过与单个ODC原聚体相互作用来抑制二聚体ODC酶活性，并且该复合物以不依赖于泛素的方式被26 S蛋白酶体降解。 虽然ODC在不同物种中高度同源，但AZ蛋白显示出广泛的多样性。哺乳动物ODC的晶体结构和大鼠抗酶的部分NMR结构是已知的。虽然ODC-AZ复合物的结构信息是未知的，但最近的计算机模拟实验已经显示了参与哺乳动物ODC-AZ结合的残基。在我们最近的研究中，我们已经从酿酒酵母中纯化并表征了ODC：AZ抑制复合物。在目前的实验中，我们提出了一个详细的研究酵母ODC：AZ异二聚体，这是很重要的了解如何酵母AZ调节ODC，并在确定参与异二聚体形成的关键残基。 我们已经纯化酵母ODC，AZ和ODC：AZ异二聚体的同质性和比较的蛋白质进行了氢氘交换，以确定接口残基是显着的异二聚体的形成。从H/D实验中鉴定了AZ中的三个肽和ODC中的四个肽，它们存在于ODC：AZ复合物的界面中。然后，我们使用系统的定点突变，以验证关键的残基AZ是重要的异源二聚体相互作用，在体内和体外试验。使用下拉测定，测试等摩尔AZ蛋白结合ODC的能力。我们观察到不同的AZ突变体对ODC具有不同的亲和力。 当对相同的样品进行AZ抑制测定时，与野生型AZ相比，各种AZ突变体显示出对ODC的抑制降低，表明这些残基的重要性。与野生型AZ相比，突变体AZ在体内显示出降低的ODC降解。对这些相同的体内样品进行了ODC活性测试，我们发现AZ突变体抑制ODC活性的能力大大降低。还分析了AZ突变体的多胺含量;与野生型相比，多胺含量增加，进一步支持AZ突变体中ODC活性降低和降解。我们的研究表明，酵母AZ含有进化保守的界面残基，这些残基对于异源二聚化是重要的，并且对于其体外和体内的结合和抑制活性是必不可少的。我们目前正在进行与BS 3（一种化学交联剂）的交联实验，然后进行质谱分析（MSMS和LCMS），以鉴定纯化的ODC：AZ异二聚体界面中AZ和ODC相互作用的残基。

Ornithine decarboxylase and antizyme interaction interface shows critical residues that are important for ornithine decarboxylase inhibition of activity and binding Ornithine decarboxylase (ODC) is a key rate limiting enzymes of polyamine biosynthesis. Polyamines are cationic molecules that play an important role in cellular proliferation, protein synthesis and gene regulation and are found in organisms ranging from bacteria to humans. In various organisms ODC activity is post-translationally regulated by its inhibitory protein antizyme (AZ), which is synthesized as a result of a ribosomal frameshifting due to increasing polyamine concentrations. AZ is expressed as a monomer, which inhibits dimeric ODC enzymatic activity by interacting with a single ODC protomer and this complex is degraded by the 26S proteasome in an ubiquitin independent manner. While ODC is highly homologous in different species, AZ protein shows a wide diversity. The crystal structure of mammalian ODC and a partial NMR structure of rat antizyme are known. Although the structural information of ODC-AZ complex is unknown, a recent in silico experiment has shown residues that are involved in mammalian ODC-AZ binding. In our recent study we have purified and characterized the ODC:AZ inhibitory complex from Saccharomyces cerevisiae. In the current experiments we present a detail study of the yeast ODC:AZ heterodimer, which are important in understanding how yeast AZ regulates ODC and in identifying the critical residues involved in heterodimer formation. We have purified yeast ODC, AZ and ODC:AZ heterodimer to homogeneity and comparisons of the proteins were performed by hydrogen deuterium exchange to identify the interface residues that are significant in forming the heterodimer. Three peptides in the AZ and four peptides in ODC were identified from H/D experiments that are present in the interface of ODC:AZ complex. We then used systematic site-directed mutagenesis to verify the critical residues of AZ that are important for heterodimer interaction in both in vivo and in vitro assays. Using a pull down assay, an equimolar AZ protein was tested for its ability to bind ODC. We observed that different AZ mutants have varying affinities to ODC. When the same samples were tested for AZ inhibition assays, various AZ mutants showed a reduced inhibition of ODC as compared to the wild type AZ showing the importance of these residues. In contrast to wild type AZ, the mutant AZ showed reduced degradation of ODC in vivo. These same in vivo samples were tested for ODC activity and we found that AZ mutants had greatly diminished ability to inhibit ODC activity. Polyamine content of the AZ mutants were also analyzed; it showed increased polyamine content compared to the wild type, further supporting the reduced activity and degradation of ODC in AZ mutants. Our study demonstrates that yeast AZ contains evolutionary conserved interface residues that are important for hetero-dimerization and are essential for its binding and inhibitory activity both in vitro and in vivo. We are currently performing a cross-linking experiment with BS3 (a chemical cross-linker) followed by mass-spectrometric analyses (MSMS and LCMS) to identify AZ and ODC interacting residues in the interface of purified ODC:AZ heterodimer.