Ridley Helen, Lakey Jeremy H
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
Microbiology (Reading). 2015 Mar;161(Pt 3):503-15. doi: 10.1099/mic.0.000024. Epub 2014 Dec 23.
Most colicins kill Escherichia coli cells by membrane pore formation or nuclease activity and, superficially, the mechanisms are similar: receptor binding, translocon recruitment, periplasmic receptor binding and membrane insertion. However, in detail, they employ a wide variety of molecular interactions that reveal a high degree of evolutionary diversification. Group A colicins bind to members of the TolQRAB complex in the periplasm and heterotrimeric complexes of colicin-TolA-TolB have been observed for both ColA and ColE9. ColN, the smallest and simplest pore-forming colicin, binds only to TolA and we show here that it uses the binding site normally used by TolB, effectively preventing formation of the larger complex used by other colicins. ColN binding to TolA was by β-strand addition with a KD of 1 µM compared with 40 µM for the TolA-TolB interaction. The β-strand addition and ColN activity could be abolished by single proline point mutations in TolA, which each removed one backbone hydrogen bond. By also blocking TolA-TolB binding these point mutations conferred a complete tol phenotype which destabilized the outer membrane, prevented both ColA and ColE9 activity, and abolished phage protein binding to TolA. These are the only point mutations known to have such pleiotropic effects and showed that the TolA-TolB β-strand addition is essential for Tol function. The formation of this simple binary ColN-TolA complex provided yet more evidence of a distinct translocation route for ColN and may help to explain the unique toxicity of its N-terminal domain.
大多数大肠杆菌素通过形成膜孔或核酸酶活性来杀死大肠杆菌细胞，表面上看，其机制相似：受体结合、转运体募集、周质受体结合和膜插入。然而，详细来说，它们采用了各种各样的分子相互作用，显示出高度的进化多样性。A组大肠杆菌素与周质中的TolQRAB复合物成员结合，并且已观察到ColA和ColE9形成大肠杆菌素-TolA-TolB异源三聚体复合物。ColN是最小且最简单的形成孔道的大肠杆菌素，仅与TolA结合，我们在此表明它使用TolB通常使用的结合位点，有效地阻止了其他大肠杆菌素使用的更大复合物的形成。ColN与TolA的结合是通过β链添加，解离常数（KD）为1 μM，而TolA-TolB相互作用的解离常数为40 μM。TolA中的单个脯氨酸点突变可消除β链添加和ColN活性，每个突变去除了一个主链氢键。通过阻断TolA-TolB结合，这些点突变赋予了完整的tol表型，使外膜不稳定，阻止了ColA和ColE9的活性，并消除了噬菌体蛋白与TolA的结合。这些是已知具有这种多效性作用的唯一的点突变，表明TolA-TolB的β链添加对于Tol功能至关重要。这种简单的二元ColN-TolA复合物的形成提供了更多证据，证明ColN存在独特的转运途径，并且可能有助于解释其N端结构域独特的毒性。
