Study on molecular structure-function relationship of E..coli DnaA protein

特定目的：DNAA蛋白对ATP/ADP有很高的亲和力，ATP结合型对DNA复制起作用，而ADP结合型不起作用。这项工作的第一个目的是在鉴定负责与ATP结合的氨基酸残基的基础上，揭示蛋白质构象变化的机制。尤其是识别ATP的伽马位置上与磷酸盐相互作用的残基，对于了解蛋白质的激活机制是至关重要的。第二个目的是确定蛋白质的结构域来调节其活性。结果和讨论：使用含有修饰磷酸的ATP类似物的亲和标记和与该类似物共价结合的氨基酸残基的测定表明，Lys-415残基与ATP的伽马磷酸发生相互作用。接下来，通过定点突变，该残基被与其他突变残基取代，并被纯化。这些分析表明，赖氨酸残基是ATP(手稿准备中)激活蛋白质所必需的。在第二个项目中，通过在Dna A顺反子中引入随机突变，我们获得了三个Dna A突变基因，它们对携带野生型等位基因的细胞具有致命性。测序表明，突变出现在DNAA蛋白的ATP结合域和DNA结合域之间的不同区域。对分离到的突变体Dna A的纯化和鉴定表明，该突变体蛋白与ATP缺乏亲和力，但仍具有复制活性。这些结果表明，这一不同区域的构象对于DNAA功能的调控具有重要意义，这是研究蛋白质结构功能关系的关键发现。

Specific aims : DnaA protein has high affinity for ATP/ADP.Tne ATP-bound form is active for initiation of DNA replication, but the ADP-bound form is not. The first purpose of this work is to reveal mechanism of conformational change of the protein, based on identification of amino acid residue responsible for the ATP-binding. Especially, identification of the residue interacting with phosphate in the gamma position of ATP is most important to know the activation mechanism of the protein. The second purpose is to identify the protein's domain to regulate its activity. For this, dnaA mutants that show lethality for cells bearing the wild-type allele, due to occurrence of excessive initiations will be isolated and biochemical analyzes of the mutant proteins will be performed.Results and Discussion : Affinity labeling using an ATP-analog that contains modified phosphate at the gamma position and determination of amino acid residue that is covalently bound to this analog suggested that Lys-415 residue interacts with the gamma phosphate of ATP.Next, by site-directed mutagenesis this residue was replace with other ones, and such mutant proteins were purified. Analyzes of these suggested that Lys residue is necessary for activation of the protein by ATP (manuscript in preparation). In the second project, by introducing random mutation in the dnaA cistron, we obtained three dnaA mutant genes that show lethality for cells-bearing the wild-type allele. Sequencing indicated that mutations appear in a distinct region between the ATP-binding domain and the DNA-binding domain of DnaA protein. Purification and characterization of a mutant DnaA isolated here revealed that this mutant protein lacks affinity for ATP but sustains replication activity. These results indicate an importance of conformation of this distinct region for regulation of DnaA function, being a critical finding in study of structure function relationship of the protein.