Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU
Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU
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DOI：
10.1099/mic.0.2007/011320-0
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发表时间：
2008-01-01
期刊：
影响因子：
2.8
通讯作者：
Burrows, Lori L.
中科院分区：
文献类型：
--
作者：
Chiang, Poney;Sampaleanu, Liliana M.;Burrows, Lori L.
IV 型菌毛是可伸缩的蛋白质纤维，被许多细菌病原体用于粘附、抽搐运动、生物膜形成和宿主定殖。在铜绿假单胞菌中，PilB 和 PilT 是属于分泌 NTPase 超家族的双极蛋白，分别驱动菌毛伸展和收缩，而单极 PilT 旁系同源物 PilU 以未知方式支持菌毛收缩。对来自铜绿假单胞菌的纯化 6xHis 标记的 PilB、PilT 和 PilU 进行的分析表明，所有三种蛋白均具有体外 ATP 酶活性。分泌 NTPase 的 Walker A (WA)、Walker B (WB)、Asp Box 和 His Box 基序中的保守残基发生突变，并测试了抽搐运动的互补性。三种 ATP 酶中任何一种的保守 WA 或 WB 残基的突变都会消除抽搐运动，并且对于 PilT 的 WA 突变体来说，会导致极性定位的丧失。 Asp Box 基序中的三个不变酸性残基和 His Box 基序中的两个不变 His 残基的要求各不相同，其中 PilB 对变化的耐受性最差。在所有三种蛋白质中，Asp 盒中的第三个酸性残基和 His 盒中的第二个 His 残基对于功能至关重要；这些残基的突变导致 PilT ATPase 体外活性丧失。这些突变对 Aquifex aeolicus PilT 和霍乱弧菌 EpsE（PilB 同源物）晶体结构影响的建模表明，关键的 Asp Box 和 His Box 残基有助于形成配体周围的催化口袋。这些结果提供了实验证据，区分对功能至关重要的广泛保守的 Asp 和 His Box 残基与其作用受特定局部环境调节的残基。
Type IV pili are retractable protein fibres used by many bacterial pathogens for adherence, twitching motility, biofilm development and host colonization. In Pseudomonas aeruginosa, PilB and PilT are bipolar proteins belonging to the secretion NTPase superfamily, and power pilus extension and retraction, respectively, while the unipolar PilT paralogue PilU supports pilus retraction in an unknown manner. Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. Conserved residues in the Walker A (WA), Walker B (WB), Asp Box and His Box motifs characteristic of secretion NTPases were mutated, and complementation of twitching motility was tested. Mutation of conserved WA or WB residues in any of the three ATPases abrogated twitching motility, and for the WA mutant of PilT caused loss of polar localization. The requirement for three invariant acidic residues in the Asp Box motif, and for two invariant His residues in the His Box motif varied, with PilB being the least tolerant of changes. In all three proteins, the third acidic residue in the Asp Box and the second His of the His Box were crucial for function; mutation of these residues caused loss of PilT ATPase activity in vitro. Modelling of the effects of these mutations on the crystal structures of Aquifex aeolicus PilT and Vibrio cholerae EpsE (a PilB homologue) showed that the critical Asp Box and His Box residues contribute to a catalytic pocket that surrounds the ligand. These results provide experimental evidence differentiating widely conserved Asp and His Box residues that are essential for function from those whose roles are modulated by specific local environments.