Nivaskumar Mangayarkarasi, Bouvier Guillaume, Campos Manuel, Nadeau Nathalie, Yu Xiong, Egelman Edward H, Nilges Michael, Francetic Olivera
Molecular Genetics Unit, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France; CNRS ERL3526, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France; University Paris VII, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France.
Structural Bioinformatics Unit, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France; CNRS UMR3528, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France.
Structure. 2014 May 6;22(5):685-96. doi: 10.1016/j.str.2014.03.001. Epub 2014 Mar 27.
The closely related bacterial type II secretion (T2S) and type IV pilus (T4P) systems are sophisticated machines that assemble dynamic fibers promoting protein transport, motility, or adhesion. Despite their essential role in virulence, the molecular mechanisms underlying helical fiber assembly remain unknown. Here, we use electron microscopy and flexible modeling to study conformational changes of PulG pili assembled by the Klebsiella oxytoca T2SS. Neural network analysis of 3,900 pilus models suggested a transition path toward low-energy conformations driven by progressive increase in fiber helical twist. Detailed predictions of interprotomer contacts along this path were tested by site-directed mutagenesis, pilus assembly, and protein secretion analyses. We demonstrate that electrostatic interactions between adjacent protomers (P-P+1) in the membrane drive pseudopilin docking, while P-P+3 and P-P+4 contacts determine downstream fiber stabilization steps. These results support a model of a spool-like assembly mechanism for fibers of the T2SS-T4P superfamily.
密切相关的细菌II型分泌（T2S）系统和IV型菌毛（T4P）系统是复杂的机器，它们组装动态纤维以促进蛋白质运输、运动或粘附。尽管它们在毒力中起着至关重要的作用，但螺旋纤维组装的分子机制仍然未知。在这里，我们使用电子显微镜和灵活建模来研究由产酸克雷伯菌T2SS组装的PulG菌毛的构象变化。对3900个菌毛模型的神经网络分析表明，随着纤维螺旋扭曲的逐渐增加，存在一条向低能量构象转变的路径。通过定点诱变、菌毛组装和蛋白质分泌分析，对沿此路径的原聚体间接触进行了详细预测。我们证明，膜中相邻原聚体（P-P+1）之间的静电相互作用驱动假菌毛对接，而P-P+3和P-P+4接触决定了下游纤维稳定步骤。这些结果支持了T2SS-T4P超家族纤维的卷轴状组装机制模型。
