The PapC usher, a β-barrel pore in the outer membrane of uropathogenic Escherichia coli, is used for assembly of the P pilus, a key virulence factor in bacterial colonization of human kidney cells. Each PapC protein is composed of a 24-stranded β-barrel channel, flanked by N- and C-terminal globular domains protruding into the periplasm, and occluded by a plug domain (PD). The PD is displaced from the channel towards the periplasm during pilus biogenesis, but the molecular mechanism for PD displacement remains unclear. Two structural features within the β-barrel, an α-helix and β5-6 hairpin loop, may play roles in controlling plug stabilization. Here we have tested clusters of residues at the interface of the plug, barrel, α-helix and hairpin, which participate in electrostatic networks. To assess the roles of these residues in plug stabilization, we used patch-clamp electrophysiology to compare the activity of wild-type and mutant PapC channels containing alanine substitutions at these sites. Mutations interrupting each of two salt bridge networks were relatively ineffective in disrupting plug stabilization. However, mutation of two pairs of arginines located at the inner and the outer surfaces of the PD resulted in an enhanced propensity for plug displacement. One arginine pair involved in a repulsive interaction between the linkers that tether the plug to the β-barrel was particularly sensitive to mutation. These results suggest that plug displacement, which is necessary for pilus assembly and translocation, may require a weakening of key electrostatic interactions between the plug linkers, and the plug and the α-helix.

PapC引入，是尿路致病性大肠杆菌外膜上的一个β桶孔，用于组装P菌毛，P菌毛是人类肾脏细胞细菌定殖的关键毒力因子。每个PapC蛋白均由24链β-桶形通道组成，两侧为突出于周质的N和C端球形结构域，并被插入结构域（PD）封闭。PD在菌毛生物发生过程中从通道向周质移位，但PD移位的分子机制仍不清楚。β-桶内的两个结构特征，即α-螺旋和β5-6发夹环，可能在控制塞子稳定中起作用。在这里，我们测试了在插头，桶，α螺旋和发夹的界面处残留的簇，它们参与了静电网络。为了评估这些残留物在塞子稳定中的作用，我们使用膜片钳电生理学来比较在这些位点包含丙氨酸取代的野生型和突变PapC通道的活性。中断两个盐桥网络中的每一个的突变在破坏塞子稳定性方面相对无效。然而，位于PD的内表面和外表面的两对精氨酸的突变导致了柱塞位移的增强倾向。一对精氨酸对参与连接子之间的排斥相互作用，该连接子将栓子束缚在β-桶上对突变特别敏感。这些结果表明，对于菌毛组装和移位而言，塞子位移是必需的，这可能需要削弱塞子接头，塞子和α-螺旋之间的关键静电相互作用。中断两个盐桥网络中的每一个的突变在破坏塞子稳定性方面相对无效。然而，位于PD的内表面和外表面的两对精氨酸的突变导致了柱塞位移的增强倾向。一对精氨酸对参与连接子之间的排斥相互作用，该连接子将栓子束缚在β-桶上对突变特别敏感。这些结果表明，对于菌毛组装和移位而言，塞子位移是必需的，这可能需要削弱塞子接头，塞子和α-螺旋之间的关键静电相互作用。中断两个盐桥网络中的每一个的突变在破坏塞子稳定性方面相对无效。然而，位于PD的内表面和外表面的两对精氨酸的突变导致了柱塞位移的增强倾向。一对精氨酸对参与连接子之间的排斥相互作用，该连接子将栓子束缚在β-桶上对突变特别敏感。这些结果表明，对于菌毛组装和移位而言，塞子位移是必需的，这可能需要削弱塞子接头，塞子和α-螺旋之间的关键静电相互作用。位于PD内表面和外表面的两对精氨酸发生突变，导致塞子位移的倾向增强。一对精氨酸对参与连接子之间的排斥相互作用，该连接子将栓子束缚在β-桶上对突变特别敏感。这些结果表明，对于菌毛组装和移位而言，塞子位移是必需的，这可能需要削弱塞子接头，塞子和α-螺旋之间的关键静电相互作用。位于PD内表面和外表面的两对精氨酸发生突变，导致塞子位移的倾向增强。一对精氨酸对参与连接子之间的排斥相互作用，该连接子将栓子束缚在β-桶上对突变特别敏感。这些结果表明，对于菌毛组装和移位而言，塞子位移是必需的，这可能需要削弱塞子接头，塞子和α-螺旋之间的关键静电相互作用。