Graduate School of Frontier Biosciences, Osaka Universitygrid.136593.b, Suita, Osaka, Japan.
School of Life Science and Technology, Tokyo Institute of Technology, Meguro, Tokyo, Japan.
Microbiol Spectr. 2022 Aug 31;10(4):e0111022. doi: 10.1128/spectrum.01110-22. Epub 2022 Jul 25.
Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhA) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhA consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhA. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side chain interaction networks in FlhA and promote the cyclic open-close domain motions of FlhA. The temperature-sensitive () mutation stabilized a completely closed conformation at 42°C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhA structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhA required for efficient and rapid protein export by the fT3SS. Many motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhA), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhA undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhA. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhA combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhA by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhA to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.
鞭毛结构亚基通过鞭毛类型 III 分泌系统 (fT3SS) 进行运输，并在生长中的鞭毛结构的远端组装。FlhA（FlhA）的 C 端细胞质结构域充当出口底物和鞭毛伴侣的对接平台，并在分层蛋白质靶向和出口中发挥重要作用。FlhA 由结构域 D1、D2、D3 和 D4 组成，并采用开放和闭合构象。沙门氏菌 FlhA 的 Gly-368 位于高度保守的 GYXLI 基序内，对 FlhA 的动态结构域运动至关重要。然而，其工作机制尚不清楚。在这里，我们报告 GYXLI 基序的周期性构象变化诱导 FlhA 中疏水性侧链相互作用网络的重塑，并促进 FlhA 的环状开-关结构域运动。温度敏感（ts）突变通过 Gln-498 与域 D1 之间以及 Pro-667 与域 D4 之间的强疏水相互作用，以及 Phe-459 与域 D2 之间和 Pro-646 之间的强疏水相互作用，在 42°C 下稳定完全关闭构象 FlhA 结构域 D4，从而抑制 fT3SS 的鞭毛蛋白输出。其基因内抑制突变重新组织了封闭 FlhA 结构中的疏水相互作用网络，在很大程度上恢复了 fT3SS 的蛋白输出活性。此外，GYXLIMotif 的构象灵活性对于鞭毛蛋白的输出至关重要。我们提出保守的 GYXLI 基序作为结构开关，诱导 FlhA 的动态结构域运动，这对于 fT3SS 高效快速的蛋白质输出是必需的。许多运动细菌利用鞭毛 III 型分泌系统 (fT3SS) 在细胞质膜外构建鞭毛。FlhA（FlhA）是 fT3SS 的跨膜亚基，其 C 端细胞质结构域为出口底物和鞭毛出口伴侣提供结合位点，以协调鞭毛蛋白的出口与组装。FlhA 经历周期性的开-关结构域运动。FlhA 的高度保守的 Gly-368 残基被假定对 FlhA 的动态结构域运动至关重要。然而，其工作机制尚不清楚。在这里，我们结合分子动力学模拟对 FlhA 进行了突变分析，并提供了证据表明 Gly-368 对 FlhA 构象灵活性的重要性在于重塑 FlhA 中的疏水性侧链相互作用网络，以促进其环状开-关结构域运动，使 fT3SS 能够有效地将鞭毛结构亚基运输到鞭毛快速组装。
