cAMP受体蛋白(CRPs)通过cAMP结合调节整体基因表达水平,在细菌环境适应中起关键作用。这里,我们报告了DdrI的结构,来自耐辐射球菌的CRP家族蛋白。结合生化,动力学,和分子动力学模拟分析,我们的结果表明,DdrI在不存在cAMP的情况下采用DNA结合构象,并且可以与经典CRPs的靶DNA序列形成稳定的复合物。进一步的分析表明,DdrI的高亲和力cAMP结合口袋部分填充了Tyr113-Arg55-Glu65侧链,模拟抗cAMP介导的变构转换。此外,与经典CRPs相比,DdrI在蛋白质-DNA界面的第二个syn-cAMP结合位点带负电,和锰离子可以增强其DNA结合亲和力。DdrI还可以与模拟另一个转录因子的靶序列结合,DdrO,提示这两种转录因子之间潜在的串扰。这些发现揭示了一类与cAMP激活无关的CRPs,并提供了对D.radiodurans环境适应机制的有价值的见解。重要细菌需要在基因转录水平上对环境变化做出反应,这对它们的进化至关重要,毒力,和工业应用。大肠杆菌(ecCRP)的cAMP受体蛋白(CRP)可感知细胞内cAMP水平的变化,是教科书中变构效应的经典例子。然而,CRPs的结构和生化活性一般不保守,存在不同的机制。在这项研究中,我们发现拟议的耐辐射球菌CRP,DdrI,表现出独立于cAMP结合的DNA结合能力,并采用类似于活化CRP的apo结构。锰可以增强DdrI的DNA结合,同时允许其靶序列具有一定程度的自由度。这些结果表明,CRP可以发展成为一类独立于cAMP的全球监管机构,使细菌能够根据其特性适应不同的环境。第一个发现的CRP家族成员,ecCRP(或CAP)很可能不是该家族的典型特征,并且与祖先的CRP家族转录因子非常不同。
The cAMP receptor proteins (CRPs) play a critical role in bacterial environmental adaptation by regulating global gene expression levels via cAMP binding. Here, we report the structure of DdrI, a CRP family protein from
Deinococcus radiodurans. Combined with biochemical, kinetic, and molecular dynamics simulations analyses, our results indicate that DdrI adopts a DNA-binding conformation in the absence of cAMP and can form stable complexes with the target DNA sequence of classical CRPs. Further analysis revealed that the high-affinity cAMP binding pocket of DdrI is partially filled with Tyr113-Arg55-Glu65 sidechains, mimicking the anti-cAMP-mediated allosteric transition. Moreover, the second syn-cAMP binding site of DdrI at the protein-DNA interface is more negatively charged compared to that of classical CRPs, and manganese ions can enhance its DNA binding affinity. DdrI can also bind to a target sequence that mimics another transcription factor, DdrO, suggesting potential cross-talk between these two transcription factors. These findings reveal a class of CRPs that are independent of cAMP activation and provide valuable insights into the environmental adaptation mechanisms of D. radiodurans.IMPORTANCEBacteria need to respond to environmental changes at the gene transcriptional level, which is critical for their evolution, virulence, and industrial applications. The cAMP receptor protein (CRP) of Escherichia coli (ecCRP) senses changes in intracellular cAMP levels and is a classic example of allosteric effects in textbooks. However, the structures and biochemical activities of CRPs are not generally conserved and there exist different mechanisms. In this study, we found that the proposed CRP from
Deinococcus radiodurans, DdrI, exhibited DNA binding ability independent of cAMP binding and adopted an apo structure resembling the activated CRP. Manganese can enhance the DNA binding of DdrI while allowing some degree of freedom for its target sequence. These results suggest that CRPs can evolve to become a class of cAMP-independent global regulators, enabling bacteria to adapt to different environments according to their characteristics. The first-discovered CRP family member, ecCRP (or CAP) may well not be typical of the family and be very different to the ancestral CRP-family transcription factor.