Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, C3BI, Institut Pasteur, CNRS UMR3528, CNRS USR3756, Paris, France.
NMR of Biomolecules Unit, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS UMR3528, Paris, France.
Structure. 2019 Jul 2;27(7):1082-1093.e5. doi: 10.1016/j.str.2019.03.021. Epub 2019 May 2.
Bacterial type 4a pili are dynamic surface filaments that promote bacterial adherence, motility, and macromolecular transport. Their genes are highly conserved among enterobacteria and their expression in enterohemorrhagic Escherichia coli (EHEC) promotes adhesion to intestinal epithelia and pro-inflammatory signaling. To define the molecular basis of EHEC pilus assembly, we determined the structure of the periplasmic domain of its major subunit PpdD (PpdDp), a prototype of an enterobacterial pilin subfamily containing two disulfide bonds. The structure of PpdDp, determined by NMR, was then docked into the density envelope of purified EHEC pili obtained by cryoelectron microscopy (cryo-EM). Cryo-EM reconstruction of EHEC pili at ∼8 Å resolution revealed extremely high pilus flexibility correlating with a large extended region of the pilin stem. Systematic mutagenesis combined with functional and interaction analyses identified charged residues essential for pilus assembly. Structural information on exposed regions and interfaces between EHEC pilins is relevant for vaccine and drug discovery.
细菌 4a 型菌毛是动态的表面丝，可促进细菌黏附、运动和大分子运输。其基因在肠杆菌中高度保守，在肠出血性大肠杆菌（EHEC）中的表达促进了对肠道上皮的黏附和促炎信号转导。为了确定 EHEC 菌毛组装的分子基础，我们测定了其主要亚基 PpdD（PpdDp）的周质结构域的结构，PpdDp 是含有两个二硫键的肠杆菌菌毛亚家族的原型。通过 NMR 确定的 PpdDp 结构，然后通过冷冻电镜（cryo-EM）获得的纯化 EHEC 菌毛的密度包埋中对接。约 8 Å 分辨率的 EHEC 菌毛的 cryo-EM 重建揭示了菌毛极高的灵活性，与菌毛干的大扩展区域相关。系统诱变与功能和相互作用分析相结合，确定了菌毛组装所必需的带电残基。EHEC 菌毛暴露区域和界面的结构信息与疫苗和药物发现有关。
