Mechanistic Studies of Functional Switching in the PutA Flavoprotein

描述（由申请人提供）：来自大肠杆菌的脯氨酸利用率A （PutA）是一种大型多功能蛋白，在单个多肽中独特地结合了酶和转录调节活性。PutA作为一种酶，通过黄素依赖的脯氨酸脱氢酶（PRODH）和nadd依赖的a1 -吡啶-5-羧酸脱氢酶（P5CDH）结构域的协同作用，与细胞质内膜外周结合，催化脯氨酸四电子氧化为谷氨酸。n端带-螺旋-螺旋基序赋予PutA与dma结合活性，使PutA也能作为脯氨酸利用（put）基因PutA和putP（编码高亲和脯氨酸转运蛋白）的胞质自生转录抑制因子。为了实现其作为转录抑制因子和膜结合脯氨酸分解代谢酶的互斥功能，PutA经历了脯氨酸依赖的功能转换。该建议的中心假设是PRODH活性位点产生的黄素氧化还原信号控制PutA的整体构象、亚细胞位置和功能。最近的研究表明，黄素辅助因子的减少驱动puta膜结合并诱导PRODH活性位点的结构变化，这一观点得到了支持。为了进一步探索这一假设，黄素辅助因子如何控制PutA功能开关的动态和结构模型将使用多种方法，包括光谱电化学，位点定向诱变，表面等离子体共振，X射线晶体学，疏水光标记和氢-氘交换质谱。本研究的主要目标是揭示PutA从基因调控蛋白转化为膜结合酶的新型氧化还原机制，并提供PutA如何在单个多肽中整合催化，膜结合和dma结合活性的结构理解。实现这一目标的具体目标如下：确定黄素蛋白相互作用，指导PutA的功能转换。2. 阐明三功能PutA的整体三维结构。3. 鉴定PutA的膜结合结构域。4. 描述脯氨酸依赖的PutA构象变化。这项工作将产生关于蛋白质如何执行多种任务的机制见解。项目成果还将进一步了解胃癌、锥虫病、I型高脯氨酸血症和精神分裂症易感性中的脯氨酸生物能量学。

DESCRIPTION (provided by applicant): Proline utilization A (PutA) from Escherichia coli is a large multifunctional protein that uniquely combines enzymatic and transcriptional regulatory activities within a single polypeptide. As an enzyme, PutA peripherally associates with the inner cytoplasmic membrane to catalyze the four-electron oxidation of proline to glutamate via the coordinated actions of separate flavin-dependent proline dehydrogenase (PRODH) and NAD-dependent A1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. An N-terminal ribbon-helix-helix motif endows PutA with DMA-binding activity enabling PutA to function also as a cytosolic autogenous transcriptional represser of the proline utilization (put) genes putA and putP (encodes a high affinity proline transporter). To fulfill its mutually exclusive functions as a transcriptional represser and membrane-bound proline catabolic enzyme, PutA undergoes proline-dependent functional switching. The central hypothesis of this proposal is that flavin redox signals generated in the PRODH active site control the global conformation, subcellular location and function of PutA. This idea is supported by recent work demonstrating that reduction of the flavin cofactor drives PutA-membrane association and induces structural changes in the PRODH active site. To further explore this hypothesis, a dynamic and structural model for how the flavin cofactor controls functional switching of PutA will be developed using a wide variety of approaches, including spectroelectrochemistry, site-directed mutagenesis, surface plasmon resonance, X- ray crystallography, hydrophobic photolabeling and hydrogen-deuterium exchange mass spectrometry. The major goals of this study are to uncover the novel redox-based mechanism whereby PutA transforms from a gene regulatory protein into a membrane-bound enzyme and to provide a structural understanding of how PutA integrates catalytic, membrane-binding and DMA-binding activities within a single polypeptide. The specific aims to achieve this are the following: 1. Identify flavin-protein interactions that direct the functional switching of PutA. 2. Elucidate the global three-dimensional architecture of trifunctional PutA. 3. Identify membrane-binding domains of PutA. 4. Characterize proline-dependent conformational changes in PutA. This work will generate mechanistic insights into how proteins perform multiple tasks. Project outcomes will also further the understanding of proline bioenergetics in gastric cancer, trypanosomal diseases, type I hyperprolinemia and schizophrenia susceptibility.