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/总结： 细菌微区室 (MCP) 是巨大的蛋白质组装体，在多种细菌中充当代谢细胞器 遍布微生物世界。这些非凡的结构由数千个组装的子单元组成 形成封装一系列顺序作用的代谢酶的多面体外壳。 MCP 通常 封装产生挥发性或有毒中间体的途径，这些中间体必须被限制并代谢为其他化合物 在扩散出 MCP 并进入细菌细胞质之前。 MCP 赋予肠道细菌特殊的生长优势 并与细菌发病机制和肠道病原体的传播有关。先前的研究主要集中于 选定的 MCP 类型，并取得了重要进展。然而，仍然存在一些机械问题 尚未得到解答，一些重要的 MCP 类型基本上没有特征。我们的双 PI 团队（Bobik 和 Yeates）主要关注沙门氏菌和其他肠道细菌使用的丙二醇利用 (Pdu) MCP 降解 1,2-丙二醇，同时隔离有毒中间体丙醛。我们在上一个周期的研究 导致了对 Pdu MCP 功能机制方面的许多重要发现和批判性见解。我们的 主要发现涵盖与蛋白质结构和组装、分子识别、分子 运输和分子进化。我们当前的提案重点关注（1）有关大会的剩余问题和 沙门氏菌 Pdu MCP 的操作，以及 (2) 对新型多样化 MCP 类别的早期研究（其中 我们通过生物信息学确定了）其关键内化酶催化基于甘氨酰自由基的反应。我们的持续 Pdu MCP 的工作将回答有关用于指导组装的蛋白质-蛋白质相互作用的突出问题 Pdu MCP——我们早期的工作发现了肽靶向序列，该序列通过以下方式指导酶封装 粘合 MCP 壳的内表面。然而，需要进一步的实验来描绘更清晰的图景 不同靶向序列的优先关联及其对高阶结构组织的贡献。在 在之前的工作中，我们还表明，Pdu MCP 中壳蛋白的孔已经进化为选择性扩散 小分子的分子运输。在我们持续的工作中，我们提出实验来研究动态和 影响 MCP 壳蛋白孔打开和关闭的蛋白质构象变化的调节。第二部分 该提案的重点是新定义且研究很少的 MCP 类别，它们封装了代谢途径 依赖于甘氨酰自由基 (Gr) 酶。在栖息于大肠和肠道的细菌中发现了许多 Gr-MCP。 可能会感染尿路。我们将开展三种拟议的 Gr 亚型的工作：一种代谢 1,2- PD（与 Pdu 系统类似，但使用不相关的酶），以及据信代谢的两种不同亚型 胆碱。我们对 Gr 系统的新研究将为理解其独特的结构和 机制。我们将回答有关它们的组成、代谢功能、组织和结构的问题。与 我们对 Pdu MCP 的研究，我们的跨学科工作将以结构和遗传学研究为指导，特别是不同的研究 壳蛋白及其孔的特性，是这些系统中分子运输现象的核心。 Pdu 和 Gr 系统也将进行比较和对比，以深入了解功能背后的原理 细菌 MCP 的多样化。