Mimori-Kiyosue Y, Vonderviszt F, Namba K
International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd., Seika, Japan.
J Mol Biol. 1997 Jul 11;270(2):222-37. doi: 10.1006/jmbi.1997.1111.
Terminal regions of flagellin, about 180 NH2 and 100 COOH-terminal residues, are well conserved and play important roles in polymerization and polymorphism of bacterial flagellar filaments. About 65 NH2 and 45 COOH-terminal residues are disordered in the monomeric form, but become folded upon filament formation. Taking advantage of the facts that relatively small segments can be cleaved off these disordered termini by limited proteolysis, and isolated fragments still form straight filaments, locations of those terminal segments have been mapped out in the filament structure by electron cryomicroscopy and helical image reconstruction. The fragments studied are F(1-486), F(20-494), F(1-461), F(30-461) and F(30-452). Regardless of the size and terminal side of truncation, the structures of the filaments reconstituted from the truncated fragments all have identical subunit packing arrangements of the Lt-type symmetry. Structural differences compared to the filament reconstituted from intact flagellin are found only around the filament axis, namely in the inner-tube region, and no obvious changes are observed in the outer-tube or the outer part of the filament. Truncation of only a few terminal residues results in misfolding of the inner-tube domains and their aggregation around the filament axis; further truncation reduces the densities of different parts of the aggregate. The filament reconstituted from F(30-461) fragment shows complete disappearance of the density corresponding to the inner-tube. When a further nine residues are removed, the spoke-like features left on the inner wall of the outer-tube become significantly smaller. Based on the structures and radial mass distributions of the filaments obtained, the previous amino acid sequence assignment to the morphological domains has been confirmed and further refined. The roles of terminal segments in the assembly regulation, and those of the double-tubular structure in the polymorphic mechanism are discussed.
鞭毛蛋白的末端区域，约180个氨基末端和100个羧基末端残基，高度保守，在细菌鞭毛丝的聚合和多态性中起重要作用。约65个氨基末端和45个羧基末端残基在单体形式下是无序的，但在丝形成时会折叠。利用有限蛋白酶解可以从这些无序末端切下相对较小片段的事实，并且分离的片段仍能形成直丝，通过电子冷冻显微镜和螺旋图像重建已在丝结构中确定了这些末端片段的位置。研究的片段有F(1-486)、F(20-494)、F(1-461)、F(30-461)和F(30-452)。无论截断的大小和末端位置如何，由截短片段重构的丝的结构都具有相同的Lt型对称亚基堆积排列。与由完整鞭毛蛋白重构的丝相比，结构差异仅出现在丝轴周围，即在内管区域，而在外管或丝的外部未观察到明显变化。仅截断少数末端残基会导致内管结构域错误折叠并在丝轴周围聚集；进一步截断会降低聚集体不同部分的密度。由F(30-461)片段重构的丝显示对应于内管的密度完全消失。当再去除九个残基时，外管内壁上留下的辐状特征会显著变小。基于所获得的丝的结构和径向质量分布，先前对形态学结构域的氨基酸序列分配已得到确认并进一步完善。讨论了末端片段在组装调控中的作用以及双管状结构在多态性机制中的作用。
