Structure-function analysis of polymorphic CDI toxin-immunity protein complexes a

描述(由申请人提供)：细菌已经进化出复杂的策略来相互竞争和交流。最近在大肠杆菌中发现了一种新的细菌间竞争机制，称为接触依赖生长抑制(CDI)。CDI系统广泛存在于各种革兰氏阴性细菌中，包括许多重要的人类病原体。CDI是由CDIB/CDIA双伙伴分泌系统介导的。CDIB是一种预测的外膜蛋白，它是CDIA外蛋白出口和组装到细胞表面所必需的。CDIA的C末端区域(CDIA-CT)含有生长抑制活性，可能被切割并转位到靶细胞中以抑制生长。CDI系统还编码CDI免疫蛋白，结合并灭活其同源CDIA-CT毒素，从而保护CDI+细胞免受自身抑制。值得注意的是，CDIA-CT的毒素结构域是多态的，相应的CdiI免疫蛋白也是高度可变的。到目前为止，我们已经确定了至少30个不同的毒素免疫家族。不同CDIA-CT/CdiI家族之间的氨基酸序列同源性通常不到20%，这强烈表明每个CDIA-CT/CdiI复合体背后的蛋白质-蛋白质相互作用是独特的。此外，我们最近发现，一些CDIA-CT结构域与特定的靶细胞蛋白相互作用，这些结合作用是激活所传递的毒素所必需的。目前，CdiI蛋白中和CDIA-CT毒素和允许因子激活CDIA-CT毒素的机制尚不清楚。我们提出了结构、生化和遗传分析，以深入了解由细菌CDI系统编码的复杂的毒素免疫网络。在这项提议中，我们要求PSI网络中心帮助解决至少十个不同的CDIA-CT/CDI络合物的晶体结构。 这一建议代表了一个独特的机会，可以阐明当毒素-免疫对在进化过程中分化时，特定结合是如何保持的。

DESCRIPTION (provided by applicant): Bacteria have evolved complex strategies to compete and communicate with one another. A new mechanism of interbacterial competition, termed contact-dependent growth inhibition (CDI) was recently discovered in Escherichia coli. CDI systems are found in a wide variety of gram-negative bacteria, including many important human pathogens. CDI is mediated by the CdiB/CdiA two-partner secretion system. CdiB is a predicted outer membrane protein that is required for the export and assembly of the CdiA exoprotein onto the cell surface. The C-terminal region of CdiA (CdiA-CT) contains the growth inhibition activity and is presumably cleaved and translocated into target cells to inhibit growth. CDI systems also encode CdiI immunity proteins, which bind and inactivate their cognate CdiA-CT toxins, thereby protecting CDI+ cells from autoinhibition. Remarkably, the CdiA-CT toxin domains are polymorphic, and accordingly their corresponding CdiI immunity proteins are also highly variable. We have identified at least 30 distinct toxin-immunity families to date. There is typically less than 20% amino acid sequence identity between different CdiA-CT/CdiI families, strongly suggesting that the protein-protein interactions underlying each CdiA-CT/CdiI complex are unique. Moreover, we have recently discovered that some CdiA-CT domains interact with specific target cell proteins termed "permissive factors", and these binding interactions are required to activate the delivered toxins. Currently, the mechanisms by which CdiI proteins neutralize and permissive factors activate CdiA-CT toxins are not understood. We propose structural, biochemical and genetic analyses to gain insights into the intricate toxin-immunity network encoded by bacterial CDI systems. In this proposal, we challenge the PSI Network center to assist in solving the crystal structures of at least ten distinct CdiA-CT/CdiI complexes. This proposal represents a unique opportunity to elucidate how specific binding is maintained as toxin-immunity pairs diverge through evolution.