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

摘要/总结： 细菌微区室（MCP）是一种巨大的蛋白质组装体，在细菌中起着代谢细胞器的作用 在微生物世界中随处可见。这些非凡的结构由数千个亚基组成， 形成一个多面体的外壳，包裹着一系列依次起作用的代谢酶。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 然后扩散出MCP并进入细菌胞质溶胶。MCP赋予肠道细菌特殊的生长优势 并且与细菌发病机理和肠道病原体的传播有关。此前的研究主要集中在 选择MCP类型，并取得了重要进展。然而，一些机械问题仍然存在， 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）主要关注丙二醇利用（Pdu）MCP，其被沙门氏菌和其他肠道细菌使用 降解1，2-丙二醇，同时螯合有毒中间体丙醛。我们在上一个周期的研究 导致了许多重要的发现和关键的见解如何Pdu MCP功能的机制方面。我们 主要发现涵盖了与蛋白质结构和组装、分子识别、分子生物学和分子生物学有关的生物学现象。 运输和分子进化。我们目前的建议侧重于（1）关于大会的剩余问题， 沙门氏菌的Pdu MCP的操作，以及（2）对新的和多样化的MCP类别的早期调查（ 我们从生物信息学上鉴定了），其关键的内在酶催化基于甘氨酰自由基的反应。我们继续 Pdu MCP的工作将回答有关蛋白质-蛋白质相互作用的悬而未决的问题，这些问题用于指导蛋白质的组装。 Pdu MCP-我们早期的工作导致了肽靶向序列的发现，这些序列通过 结合MCP壳的内表面。然而，还需要进一步的实验来更清楚地描绘 不同靶向序列的优先关联及其对高阶结构组织的贡献。在 在先前的工作中，我们还表明，通过Pdu MCP中的壳蛋白的孔已经进化为选择性扩散 小分子的分子运输。在我们继续的工作中，我们提出了实验来研究动力学， 调节影响MCP壳蛋白中孔打开和关闭的蛋白质构象变化。第二部分 该提案的重点是新定义的和很少研究的一类MCP，封装代谢途径 依赖于甘氨酰自由基（Gr）酶。许多Gr-MCP存在于大肠中的细菌中， 会感染泌尿道我们将对三种提出的Gr亚型进行研究：一种是代谢1，2- PD（类似于Pdu系统，但使用不相关的酶），以及两种不同的亚型，据信代谢 胆碱我们对Gr系统的新研究将为理解其独特的结构奠定基础， 机制等我们将回答有关其组成，代谢功能，组织和结构的问题。如同 我们对Pdu MCP的研究，我们的跨学科工作将以结构和遗传研究为指导，特别是对不同的 壳蛋白及其孔的性质，这是在这些系统中的分子传输现象的核心。 Pdu和Gr系统也将进行比较和对比，以深入了解功能的基本原理 细菌MCP的多样性。

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.