Housden Nicholas G, Rassam Patrice, Lee Sejeong, Samsudin Firdaus, Kaminska Renata, Sharp Connor, Goult Jonathan D, Francis Marie-Louise, Khalid Syma, Bayley Hagan, Kleanthous Colin
Department of Biochemistry , University of Oxford , South Parks Road , Oxford OX1 3QU , U.K.
Department of Chemistry , University of Oxford , 12 Mansfield Road , Oxford OX1 3TA , U.K.
Biochemistry. 2018 Jul 24;57(29):4374-4381. doi: 10.1021/acs.biochem.8b00621. Epub 2018 Jul 6.
Protein bacteriocins are potent narrow spectrum antibiotics that exploit outer membrane porins to kill bacteria by poorly understood mechanisms. Here, we determine how colicins, bacteriocins specific for Escherichia coli, engage the trimeric porin OmpF to initiate toxin entry. The N-terminal ∼80 residues of the nuclease colicin ColE9 are intrinsically unstructured and house two OmpF binding sites (OBS1 and OBS2) that reside within the pores of OmpF and which flank an epitope that binds periplasmic TolB. Using a combination of molecular dynamics simulations, chemical trimerization, isothermal titration calorimetry, fluorescence microscopy, and single channel recording planar lipid bilayer measurements, we show that this arrangement is achieved by OBS2 binding from the extracellular face of OmpF, while the interaction of OBS1 occurs from the periplasmic face of OmpF. Our study shows how the narrow pores of oligomeric porins are exploited by colicin disordered regions for direction-specific binding, which ensures the constrained presentation of an activating signal within the bacterial periplasm.
蛋白质细菌素是一类强效窄谱抗生素，它们通过尚不清楚的机制利用外膜孔蛋白来杀死细菌。在此，我们确定了大肠杆菌特异性细菌素——大肠杆菌素如何与三聚体孔蛋白OmpF结合以启动毒素进入。核酸酶大肠杆菌素ColE9的N端约80个残基本质上是无序的，包含两个OmpF结合位点（OBS1和OBS2），它们位于OmpF的孔内，且位于一个结合周质TolB的表位两侧。通过结合分子动力学模拟、化学三聚化、等温滴定量热法、荧光显微镜和单通道记录平面脂质双层测量，我们表明这种排列是通过OBS2从OmpF的细胞外表面结合实现的，而OBS1的相互作用则发生在OmpF的周质表面。我们的研究表明，大肠杆菌素无序区域如何利用寡聚孔蛋白的狭窄孔进行方向特异性结合，这确保了在细菌周质内激活信号的受限呈现。
