Allosteric signalling in the outer membrane translocation domain of PapC usher.
Allosteric signalling in the outer membrane translocation domain of PapC usher.
复制标题
PAPC USHER的外膜易位域中的变构信号传导。
作者：
Farabella I;Pham T;Henderson NS;Geibel S;Phan G;Thanassi DG;Delcour AH;Waksman G;Topf M
PapC ushers是一种外膜蛋白，可使P皮利在尿路致病性大肠杆菌中组装和分泌。杆菌它们的转运结构域是一个被塞子结构域封闭的大β桶，塞子结构域被置换以允许菌毛亚基跨膜转运。以前的研究表明，这种门控机制是由β-发夹和α-螺旋控制的。为了研究这些元件在变构信号通讯中的作用，我们开发了一种结合天然易位结构域和缺乏β-发夹和/或α-螺旋的突变体的进化和分子动力学研究的方法。对杂合残基相互作用网络的分析表明，易位结构域（特别是β12-β14周围）内的不同区域（残基“社区”）连接这些元件，从而调节PapC门控。一组丙氨酸取代突变体的抗生素敏感性和电生理学实验证实了这些社区的功能作用。本研究阐明了PapC ushers的门控机制及其在维持外膜通透性中的重要性。DOI：http://dx.doi.org/10.7554/eLife.03532.001大肠杆菌是一种细菌，通常生活在包括人类在内的哺乳动物的肠道中，在那里它通常是无害的，甚至可以对其宿主有益。然而，某些类型的E.大肠杆菌产生毛发状的丝状物，称为P皮利，使细菌附着在人体泌尿道上并引起疾病。穿过E.在大肠杆菌细胞中，纤维必须穿过膜上一种叫做PapC usher的蛋白质。PapC usher蛋白也参与了P皮利的组装，它包含一个称为β-桶的管状部分，通常被蛋白质的另一个称为“塞子结构域”的部分阻断。为了使P皮利穿过β-桶，塞结构域必须移动。这种运动由PapC蛋白的两个部分控制，称为α-螺旋和β-发夹，但目前尚不清楚如何控制。为了解决这个问题，Farabella等人制作了正常PapC蛋白和缺乏α-螺旋和/或β-发夹的版本的计算机模型。观察这些结构模型并分析PapC蛋白的进化有助于预测β-桶的某些区域可能参与控制塞子结构域的运动，然后通过实验证实了这一点。Farabella等人提出，这些区域与α-螺旋和β-发夹一起控制β-桶的打开和关闭。需要进一步的工作来研究PapC蛋白的其他部分如何参与P皮利的形成。这些新的见解可能有助于开发对抗细菌感染的替代治疗方法。DOI：http://dx.doi.org/10.7554/eLife.03532.002网站
PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue ‘communities’) within the translocation domain (especially around β12–β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability. DOI: http://dx.doi.org/10.7554/eLife.03532.001 Escherichia coli is a bacterium that commonly lives in the intestines of mammals, including humans, where it is usually harmless and can even be beneficial to its host. However, some types of E. coli produce hair-like filaments called P pili that allow the bacteria to attach to the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, the filaments have to travel through a protein in the membrane called PapC usher. The PapC usher protein—which is also involved in the assembly of the P pili filaments—contains a tube-like part called a β-barrel that is usually blocked by another part of the protein called the ‘plug domain’. For the P pili to pass through the β-barrel, the plug domain has to move. This movement is controlled by two parts of the PapC protein, known as the α-helix and the β-hairpin, but it is not clear how. To address this question, Farabella et al. made computer models of the normal PapC protein and versions that lacked the α-helix and/or the β-hairpin. Looking at these structural models and analyzing the evolution of PapC proteins helped to predict that certain regions of the β-barrel may be involved in controlling the movement of the plug domain, and this was then confirmed experimentally. Farabella et al. propose that these regions—together with the α-helix and β-hairpin—control the opening and closing of the β-barrel. Further work is needed to investigate how other parts of the PapC protein are involved in P pili formation. These new insights could prove useful in the development of alternative treatments to fight bacterial infection. DOI: http://dx.doi.org/10.7554/eLife.03532.002