Dokas Stephen P, Taylor Daniel K, Good Lydia L, Mohanaraj Sanuja, Maillard Rodrigo A
Department of Chemistry, Georgetown University, Washington, District of Columbia 20057, United States.
Institute of Soft Matter Synthesis and Metrology, Georgetown University, Washington, District of Columbia 20057, United States.
Biochemistry. 2025 Feb 18;64(4):801-811. doi: 10.1021/acs.biochem.4c00723. Epub 2025 Jan 31.
Understanding the mechanisms of allosteric regulation in response to second messengers is crucial for advancing basic and applied research. This study focuses on the differential allosteric regulation by the ubiquitous signaling molecule, cAMP, in the cAMP receptor protein from (CRP) and from (CRP). By introducing structurally homologous mutations from allosteric hotspots previously identified in CRP into CRP and examining their effects on protein solution structure, stability and function, we aimed to determine the factors contributing to their differential allosteric regulation. Our results demonstrate that the mutations did not significantly alter the overall fold, assembly and thermodynamic stability of CRP, but had varying effects on cAMP binding affinity and cooperativity. Interestingly, the mutations had minimal impact on the specific binding of CRP to DNA promoter sites. However, we found that cAMP primarily reduces nonspecific CRP-DNA complexes and that the mutants largely lose this ability. Furthermore, our experiments revealed that CRP-DNA complexes serve as a nucleation point for additional binding of CRP proteins to form high-order oligomers with the DNA. Overall, our findings highlight the importance of both cAMP and DNA interactions in modulating the allosteric regulation of CRP and provide insights into the differential responses of CRP and CRP to cAMP.
了解变构调节响应第二信使的机制对于推进基础研究和应用研究至关重要。本研究聚焦于普遍存在的信号分子cAMP对来自[具体来源1]的cAMP受体蛋白（CRP）和来自[具体来源2]的CRP的差异变构调节。通过将先前在CRP中鉴定出的变构热点的结构同源突变引入CRP，并研究它们对蛋白质溶液结构、稳定性和功能的影响，我们旨在确定导致它们差异变构调节的因素。我们的结果表明，这些突变并未显著改变CRP的整体折叠、组装和热力学稳定性，但对cAMP结合亲和力和协同性有不同影响。有趣的是，这些突变对CRP与DNA启动子位点的特异性结合影响极小。然而，我们发现cAMP主要减少非特异性CRP-DNA复合物，而突变体在很大程度上丧失了这种能力。此外，我们的实验表明，CRP-DNA复合物作为CRP蛋白额外结合的成核点，与DNA形成高阶寡聚体。总体而言，我们的研究结果突出了cAMP和DNA相互作用在调节CRP变构调节中的重要性，并为CRP和CRP对cAMP的差异反应提供了见解。
