Vonderviszt F, Imada K, Furukawa Y, Uedaira H, Taniguchi H, Namba K
International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd, 3-4 Hikaridai, Seika, 619-0237, Japan.
J Mol Biol. 1998 Dec 18;284(5):1399-416. doi: 10.1006/jmbi.1998.2274.
HAP2 forms a capping structure, which binds very tightly to the distal end of flagellar filaments and still allows insertion of flagellin subunits below the cap by an unknown mechanism. Terminal regions of HAP2 from Salmonella typhimurium were found to be quickly degraded by various proteases, indicating that HAP2 also possesses disordered terminal regions like other axial proteins of bacterial flagellum. Removal of these portions by trypsin results in a fragment of 40 kDa (HP40), which lacks 42 NH2-terminal and 51 COOH-terminal residues. HAP2 in solution readily associates into a decameric structure without any significant population of intermediate oligomeric forms. The HP40 fragments, however, do not form decamers, while they can assemble into pentamers, as revealed by chemical cross-linking and analytical ultracentrifugation. Decameric HAP2 also dissociates into pentamers and smaller oligomers upon a heat induced conformational transition around 36 degreesC. While the highly mobile terminal regions are immobilized in decameric HAP2 complexes, they are still largely disordered in the pentameric state. These results demonstrate that the intersubunit interactions within the pentamers are mainly through the HP40 portions, whereas the terminal regions are responsible for association of pentamers into decameric complexes. Several observations indicate that HAP2 performs its capping function as a pentamer. We suggest that binding of the pentameric HAP2 cap to the filament is mediated by the highly flexible terminal regions. Indeed, HP40 fragments are unable to cap the end of filaments, while removal of about 30 residues from both terminal regions of HAP2 results in a highly reduced capping ability. A model is presented to explain the molecular mechanism of capping, in which conformational entropy in the disordered terminal regions moderates the otherwise too tight HAP2-filament interactions to allow insertion of flagellin subunits below the cap.
HAP2形成一种帽状结构，它与鞭毛丝的远端紧密结合，并且仍能通过未知机制使鞭毛蛋白亚基在帽下方插入。发现鼠伤寒沙门氏菌的HAP2末端区域会被各种蛋白酶迅速降解，这表明HAP2也像细菌鞭毛的其他轴向蛋白一样具有无序的末端区域。用胰蛋白酶去除这些部分会产生一个40 kDa的片段（HP40），它缺少42个氨基末端和51个羧基末端残基。溶液中的HAP2很容易缔合成十聚体结构，没有任何明显的中间寡聚体形式。然而，化学交联和分析超速离心显示，HP40片段不会形成十聚体，而能组装成五聚体。十聚体HAP2在36℃左右的热诱导构象转变时也会解离成五聚体和更小的寡聚体。虽然高度可移动的末端区域在十聚体HAP2复合物中被固定，但在五聚体状态下它们仍然很大程度上是无序的。这些结果表明，五聚体内的亚基间相互作用主要通过HP40部分，而末端区域负责五聚体缔合成十聚体复合物。一些观察结果表明，HAP2作为五聚体发挥其帽化功能。我们认为五聚体HAP2帽与丝状体的结合是由高度灵活的末端区域介导的。实际上，HP40片段无法封盖丝状体的末端，而从HAP2的两个末端区域去除约30个残基会导致帽化能力大幅降低。本文提出了一个模型来解释帽化的分子机制，其中无序末端区域的构象熵调节了原本过于紧密的HAP2 - 丝状体相互作用，从而允许鞭毛蛋白亚基在帽下方插入。
