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

摘要/摘要： 细菌微室(MCP)是在不同细菌中作为代谢细胞器的巨型蛋白质集合 在微生物世界中随处可见。这些非同寻常的结构由成千上万的亚基组成，这些亚基组装在一起 形成包裹一系列顺序作用的代谢酶的多面体外壳。MCPS通常 包裹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成其他化合物 在扩散出MCP并进入细菌胞质之前。MCPs赋予肠道细菌特殊的生长优势 并与细菌的发病机制和肠道病原体的传播有关。以前的研究主要集中在 关于选定的MCP类型，已经取得了重要进展。然而，一些机械性的问题仍然存在。 未回答和一些重要的MCP类型本质上是未描述的。我们的双PI团队之前的工作(Bobik和 Yates)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。我们在上一个周期中的研究 导致了许多重要的发现和对PDU MCP如何工作的机械方面的批判性见解。我们的 主要发现包括与蛋白质结构和组装、分子识别、分子 运输和分子进化。我们目前的提案集中于(1)关于大会的剩余问题和 沙门氏菌PDU监控中心的运作；及(2)对一类新的及不同类别的监控中心( 我们通过生物信息学鉴定)，其关键的内化酶催化基于甘氨酰的反应。我们的继续 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 PDU MCP-我们早期的工作导致了多肽靶向序列的发现，这些序列通过 结合MCP外壳的内表面。然而，还需要进一步的实验才能更清楚地描绘出 不同靶向序列的优先关联及其对更高层次结构组织的贡献。在……里面 在之前的工作中，我们还表明，通过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。在我们继续的工作中，我们提议进行实验来研究动力学和 MCP壳蛋白中影响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的、很少研究的一类MCP，它们封装了代谢途径 依赖于甘氨酰自由基(Gr)酶。在大肠和大肠中的细菌中发现了许多Gr-MCP 会感染尿路的细菌。我们将对三种拟议的GR亚型进行研究：一种类型代谢1，2- PD(类似于PDU系统，但使用无关的酶)和两个被认为可以代谢的不同亚型 胆碱。我们对GR系统的新研究将为理解其独特的结构和 机械装置。我们将回答有关它们的组成、代谢功能、组织和结构的问题。和以前一样 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 壳蛋白及其毛孔的性质，这是这些系统中分子传输现象的核心。 还将对PDU和GR系统进行比较和对比，以深入了解作为功能基础的原则 细菌MCP的多样化。

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.