Abstract：
Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.
摘要：
转录通过RNA聚合酶进行,并且通过与转录因子的一系列相互作用来调节。代谢物激活剂阻遏剂（Cra），LacI家族转录因子调节肠出血性大肠杆菌（EHEC）中的毒力基因表达，因此是发现抗毒力分子的有希望的药物靶标。这里,我们报道了来自大肠杆菌（EcCra）的与HEPES分子复合的Cra效应分子结合域的晶体结构。基于EcCra-HEPES复合物结构,进行配体筛选，鉴定suliosbenzone为EcCra的潜在抑制剂。电泳迁移率变化测定（EMSA）和体外转录测定验证了suliosbenzone与EcCra的结合。此外，等温滴定量热法（ITC）实验表明，与HEPES分子相比，舒利苯松（KD360nM）的结合亲和力高40倍。最后,确定了suliosbenzone结合的EcCra复合物晶体结构，以阐明suliosbenzone与EcCra的效应子结合袋的结合机制。一起,这项研究表明，舒利索苯宗可能是一个有希望的候选药物，可以作为一种有效的抗EHEC的抗毒剂进行研究和开发。
