Department of Infectious Disease, Imperial College, London, UK.
Department of Molecular Microbiology, John Innes Centre, Norwich, UK.
Nat Commun. 2024 Aug 5;15(1):6635. doi: 10.1038/s41467-024-50280-6.
The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.
细菌紧密附着菌毛系统（TadPS）组装表面菌毛，对于许多人类病原体的粘附和定植至关重要。菌毛动力学由 ATP 酶 CpaF（TadA）提供动力，通过未知的机制在新月柄杆菌中驱动延伸和缩回循环。在这里，我们使用低温电子显微镜和基于细胞的荧光显微镜来描述 CpaF 的机制。我们表明，CpaF 在不同核苷酸状态下组装成具有 C2 对称性的六聚体。核苷酸循环通过一种亚基内夹式机制发生，该机制促进亚基之间的顺序构象变化。此外，与结合不同核苷酸的活性位点进行比较表明了一种双向运动的机制。预测与平台蛋白 CpaG（TadB）和 CpaH（TadC）相互作用的保守 CpaF 残基在体内发生突变，以确定它们在菌毛加工中的作用。我们的发现为 CpaF 如何驱动 TadPS 菌毛动力学提供了模型，并对其他古老的 4 型丝状体家族成员如何为菌毛组装提供动力具有广泛的影响。
