Dissecting the structure, function, and mechanisms of diverse protein-based metabolic organelles in bacteria

剖析细菌中多种基于蛋白质的代谢细胞器的结构、功能和机制

• 批准号: 9916683
• 负责人: THOMAS Aquinas BOBIK
• 金额: $ 58.82万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916683
• 关键词: Affect; Allosteric Site; Bacteria; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Phenomena; C-terminal; Capsid; Cells; Choline; Complex; Crystallization; Cytosol; Data; Diffuse; Dissection; Encapsulated; Enterobacteriaceae; Enzymes; Evolution; Exposure to; Family; Foundations; Genes; Genetic study; Growth; Heart; Heart Diseases; Higher Order Chromatin Structure; Human; Investigation; Large Intestine; Lead; Link; Metabolic; Metabolic Pathway; Methods; Modeling; Molecular Evolution; Movement; Mutagenesis; N-terminal; Operon; Organelles; Paint; Pathogenesis; Pathway interactions; Peptides; Physiological; Play; Production; Property; Propylene Glycols; Protein Conformation; Protein Subunits; Proteins; Reaction; Regulation; Research; Role; Salmonella; Series; Structure; Surface; System; Tail; Testing; Toxic effect; Translating; Transport Process; Urinary tract; Ursidae Family; Work; bacterial metabolism; base; biological systems; biophysical analysis; biophysical techniques; cofactor; comparative genomics; enteric pathogen; enzyme activity; experimental study; in vivo; insight; microbial; molecular dynamics; molecular recognition; mutant; operation; organizational structure; pathogenic bacteria; protein protein interaction; protein structure; small molecule

Abstract/Summary: Bacterial microcompartments (MCPs) are giant protein assemblies that serve as metabolic organelles in diverse bacteria found throughout the microbial world. These extraordinary structures are composed of thousands of subunits that assemble to form a polyhedral outer shell encapsulating a series of sequentially acting metabolic enzymes. MCPs typically encapsulate pathways that produce volatile or toxic intermediates that must be confined and metabolized to other compounds before diffusing out of the MCP and into the bacterial cytosol. MCPs confer special growth advantages to enteric bacteria and are linked to bacterial pathogenesis and the dissemination of enteric pathogens. Prior studies have focused primarily on selected MCP types, and important advances have been made. However, a number of mechanistic questions remain unanswered and some important MCP types are essentially uncharacterized. Prior work by our dual-PI team (Bobik and Yeates) focused primarily on the propanediol utilization (Pdu) MCP, which is used by Salmonella and other enteric bacteria to degrade 1,2-propanediol while sequestering a toxic intermediate, propionaldehyde. Our research in the previous cycle led to numerous important discoveries and critical insights into mechanistic aspects of how the Pdu MCP functions. Our key findings cover biological phenomena related to protein structure and assembly, molecular recognition, molecular transport, and molecular evolution. Our current proposal focuses on (1) remaining questions about the assembly and operation of the Pdu MCP of Salmonella, and (2) early-stage investigations into a new and diverse class of MCPs (which we identified bioinformatically) whose key internalized enzymes catalyze glycyl-radical-based reactions. Our continuing work on the Pdu MCP will answer outstanding questions about protein-protein interactions used to guide the assembly of the Pdu MCP– our earlier work led to the discovery of peptide targeting sequences that direct enzyme encapsulation by binding the interior surface of MCP shells. However, further experiments are required to paint a clearer picture about preferential associations by varied targeting sequences and their contribution to higher-order structural organization. In prior work, we also showed that pores through the shell proteins in the Pdu MCP have evolved for selective diffusive molecular transport of small molecules. In our continuing work, we propose experiments to investigate the dynamics and regulation of protein conformational changes that affect pore opening and closing in MCP shell proteins. The second part of the proposal focuses on the newly-defined and little-studied class of MCPs that encapsulate metabolic pathways dependent on glycyl-radical (Gr) enzymes. A number of Gr-MCPs are found in bacteria that inhabit the large intestine and which can infect the urinary tract. We will undertake work on three proposed Gr subtypes: one type that metabolizes 1,2- PD (similarly to the Pdu system but using unrelated enzymes), and two distinct subtypes that are believed to metabolize choline. Our new research on the Gr systems will lay the foundations for understanding their unique structures and mechanisms. We will answer questions about their composition, metabolic function, organization, and structure. As with our studies of the Pdu MCP, our interdisciplinary work will be guided by structural and genetic studies, especially of diverse shell proteins and the properties of their pores, which are at the heart of molecular transport phenomena in these systems. The Pdu and Gr systems will also be compared and contrasted to gain insights into the principles that underlie functional diversification of bacterial MCPs.

摘要/摘要： 细菌微室（MCP）是巨型蛋白质组件，作为潜水细菌的代谢细胞器 在整个微生物世界中发现。这些非凡的结构由成千上万的亚基组成 形成一个封装一系列顺序作用代谢酶的多面体外壳。通常是MCP 封装产生挥发性或有毒中间体的途径，必须限制并代谢到其他化合物 从MCP扩散并进入细菌细胞质之前。 MCP赋予特殊增长优势进入细菌 并与细菌的发病机理和促进病原体的传播有关。先前的研究重点是主要 在选定的MCP类型上，已经取得了重要的进步。但是，仍然存在许多机械问题 未回答和一些重要的MCP类型本质上是未经表征的。我们的双PI团队的事先工作（Bobik和 Yeates）主要专注于丙二醇利用率（PDU）MCP，沙门氏菌和其他肠细菌使用 在隔离有毒的中间丙醛的同时，降解1,2-丙二醇。我们在上一个周期中的研究 导致了许多重要的发现和对PDU MCP运作方式的机理方面的重要见解。我们的 主要发现涵盖了与蛋白质结构和组装，分子识别，分子相关的生物学现象 运输和分子进化。我们当前的建议重点是（1）有关大会的剩余问题， 沙门氏菌PDU MCP的操作，以及（2）对新的和潜水员类的MCPS的早期调查（ 我们从生物信息上鉴定出其关键内在化酶催化基于糖基的反应。我们继续 PDU MCP上的工作将回答有关用于指导组装的蛋白质 - 蛋白质相互作用的杰出问题 PDU MCP - 我们较早的工作导致发现了通过酶封装的辣椒靶向序列 结合MCP壳的内部表面。但是，需要进一步的实验才能描绘出更清晰的图片 通过各种靶向序列及其对高阶结构组织的贡献来优先关联。在 先前的工作，我们还表明，通过PDU MCP中壳蛋白的孔已经进化以进行选择性扩散 小分子的分子转运。在我们的持续工作中，我们提出了实验，以研究动态和 调节蛋白质构象变化影响MCP壳蛋白中孔口开口和闭合的调节。第二部分 该提案的重点是封装新陈代谢途径的新定义和研究较少的MCP 取决于甘酰基 - 自由基（GR）酶。在细菌中发现了许多GR-MCP，这些GR居住在大肠和 会感染尿路。我们将对三种拟议的GR亚型进行工作：一种代谢1,2-- PD（类似于PDU系统，但使用无关酶）和两个不同的亚型，它们被认为代谢 胆碱。我们对GR系统的新研究将奠定基础，以了解其独特的结构和 机制。我们将回答有关其组成，代谢功能，组织和结构的问题。和 我们对PDU MCP的研究，我们的跨学科工作将由结构和遗传研究，尤其是潜水员的指导 壳蛋白及其毛孔的特性，它们是这些系统中分子转运现象的核心。 PDU和GR系统也将被比较并形成鲜明对比，以洞悉其功能基础的原则 细菌MCP的多样化。