Bio-Nanosystems Laboratory, Faculty of Information Technology, Research Institute of Chemical and Process Engineering, University of Pannonia, Egyetem u 10, H-8200 Veszprém, Hungary.
J Mol Biol. 2010 Nov 5;403(4):607-15. doi: 10.1016/j.jmb.2010.09.024. Epub 2010 Sep 22.
The hypervariable D3 domain of Salmonella flagellin, composed of residues 190-283, is situated at the outer surface of flagellar filaments. A flagellin mutant deprived of the complete D3 domain (ΔD3_FliC) exhibited a significantly decreased thermal stability (T(m) 41.9 °C) as compared to intact flagellin (T(m) 47.3 °C). However, the stability of filaments formed from ΔD3_FliC subunits was virtually identical with that of native flagellar filaments. While D3 comprises the most stable part of monomeric flagellin playing an important role in the stabilization of the other two (D1 and D2) domains, the situation is reversed in the polymeric state. Upon filament formation, ordering of the disordered terminal regions of flagellin in the core part of the filament results in the stabilization of the radially arranged D1 and D2 domains, and there is a substantial increase of stability even in the distant outermost D3 domain, which is connected to D2 via a pair of short antiparallel β-strands. Our experiments revealed that crosslinking the ends of the isolated D3 domain through a disulfide bridge gives rise to a stabilization effect reminiscent of that observed upon polymerization. It appears that the short interdomain linker between domains D2 and D3 serves as a stabilization center that facilitates propagation of the conformational signal from the filament core to the outer part of filament. Because D3 is a largely independent part of flagellin, its replacement by heterologous proteins or domains might offer a promising approach for creation of various fusion proteins possessing polymerization ability.
沙门氏菌鞭毛蛋白的超变区 D3 结构域由 190-283 位残基组成，位于鞭毛丝的外表面。与完整的鞭毛蛋白（Tm47.3°C）相比，缺失完整 D3 结构域的鞭毛蛋白突变体（ΔD3_FliC）表现出明显降低的热稳定性（Tm41.9°C）。然而，由ΔD3_FliC 亚基形成的丝状体的稳定性与天然鞭毛丝的稳定性几乎相同。虽然 D3 是单体鞭毛蛋白中最稳定的部分，对其他两个（D1 和 D2）结构域的稳定起着重要作用，但在聚合状态下情况正好相反。在丝状体形成时，无序的鞭毛蛋白末端区域在丝状体的核心部分进行排序，导致径向排列的 D1 和 D2 结构域稳定，即使在连接 D2 的最远处的最外 D3 结构域也会有很大的稳定性增加，这是通过一对短的反平行β-链连接的。我们的实验表明，通过二硫键交联分离的 D3 结构域的末端会产生类似于聚合时观察到的稳定化效应。似乎 D2 和 D3 结构域之间的短结构域连接体充当稳定中心，有助于将构象信号从丝状体核心传递到丝状体的外部。因为 D3 是鞭毛蛋白的一个很大程度上独立的部分，所以用异源蛋白或结构域替代它可能为创建具有聚合能力的各种融合蛋白提供一种很有前途的方法。
