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A(PUTA)是一种大型多功能蛋白质，它在单一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(PROH)和NAD依赖的A1-吡咯烷-5-羧酸脱氢酶(P5CDH)结构域的协同作用，与细胞内质膜结合，催化四电子氧化合成谷氨酸。一个N-末端的带状-螺旋-螺旋基序赋予putA与DMA结合的活性，使putA也能作为一个胞质内的自体转录抑制因子，抑制脯氨酸利用(PutA)基因putA和putP(编码一个高亲和力的脯氨酸转运体)。PUTA作为转录抑制因子和膜结合的Pro分解代谢酶，为了完成其相互排斥的功能，它经历了依赖于Pro的功能转换。这一假设的中心假设是，在PROSH活性部位产生的黄素氧化还原信号控制着PuTA的整体构象、亚细胞位置和功能。这一观点得到了最近工作的支持，该工作表明，黄素辅因子的减少推动了膜-膜结合，并诱导了PROSH活性部位的结构变化。为了进一步探索这一假说，将使用多种方法建立黄素辅助因子如何控制PuTA功能切换的动态和结构模型，包括光谱电化学、定点突变、表面等离子体共振、X射线结晶学、疏水光标记和氢-重离子交换质谱学。这项研究的主要目的是揭示PUTA从基因调控蛋白转化为膜结合酶的新的氧化还原机制，并提供对PUTA如何在单一多肽中整合催化、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：1.确定指导Puta功能切换的黄素-蛋白质相互作用。2.阐明了三功能PUTA的全球三维结构。3.鉴定PUTA的膜结合区。4.研究PUTA中依赖于脯氨酸的构象变化。这项工作将产生对蛋白质如何执行多项任务的机械性见解。该项目的成果还将进一步加深对脯氨酸生物能量学在胃癌、锥虫疾病、I型高脯氨酸血症和精神分裂症易感性方面的理解。

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.