PDF(Pubmed)
Abstract：
In bacterial flagellum biogenesis, secretion of the hook-filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT, nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homolog of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α-helices and resembles the FliT chaperone, sharing a similar substrate-binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126-144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size-exclusion chromatography and multi-angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled-coil interface between the third helix of FlgN and the C-terminal helix of the substrate. A FlgNΔ126-144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126-144 , like FliT, binds with sub-micromolar affinity to the flagellum ATPase FliI or its N-terminal domain. Hence FlgN and FliT likely couple delivery of low-abundance export substrates to the flagellum ATPase FliI.
摘要：
在细菌鞭毛生物发生中，钩-丝连接蛋白FlgK和FlgL的分泌和鞭毛的完成需要FlgN伴侣。同样，相关的FliT伴侣对于丝帽蛋白FliD的分泌是必需的，并结合鞭毛出口门蛋白FlhA和鞭毛ATPaseFliI。FlgN和FliT需要FliJ才能有效分泌底物。在幽门螺杆菌中，也不是FlgN,FliT和FliJ都有注释。我们证明HP1120的基因组位置与其他鞭毛细菌中的flgN相同，并且HP1120是空肠弯曲杆菌FlgN的同源物。建模的HP1120结构包含三个α螺旋，类似于FliT伴侣，共享一个类似的底物结合袋。使用下拉和热泳，我们显示HP1120和HP1120Δ126-144缺失突变体以纳摩尔亲和力与FlgK结合，但不是丝帽蛋白FliD，确认HP1120为FlgN。基于尺寸排阻色谱和多角度光散射，幽门螺杆菌FlgN以1:1化学计量与FlgK结合。FlgN和FliT之间的总体结构相似性表明，FlgN上的底物识别主要涉及FlgN的第三螺旋与底物的C末端螺旋之间的反平行卷曲螺旋界面。AFlgNΔ126-144N100A,Y103A,靶向该界面的S111I三重突变体显著损害FlgK的结合。最后,我们证明了FlgNΔ126-144，像Flit,以亚微摩尔亲和力与鞭毛ATPaseFliI或其N末端结构域结合。因此，FlgN和FliT可能将低丰度出口底物的递送耦合到鞭毛ATPaseFliI。本文受版权保护。保留所有权利。
