Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum.
Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum.
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鱼类病原体鳗弧菌 TonB2 蛋白的分子特征。
DOI：
10.1042/bj20081462
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发表时间：
2009-02-15
期刊：
影响因子：
--
通讯作者：
Vogel HJ
中科院分区：
文献类型：
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作者：
López CS;Peacock RS;Crosa JH;Vogel HJ
在鱼类病原体鳗弧菌中，TonB 2蛋白对于吸收固有的铁载体鳗毒素是必需的。在这里，我们描述的缺失突变体和丙氨酸取代影响最后6个氨基酸的TonB 2。TonB 2 C-末端两个以上氨基酸的缺失废除了铁-anguibactin运输，而最后三个残基的替换导致具有野生型运输特性的蛋白质。我们已经解决了高分辨率的溶液结构的TonB 2的C-末端域的NMR光谱。该结构域的核心（残基121-206）具有αββαβ结构，而残基76-120是柔性的和延伸的。这种整体折叠拓扑结构类似于大肠杆菌TonB C-末端结构域，尽管有两个差异：在TonB C-末端发现的β4链在TonB 2中不存在，并且环3在TonB 2中延伸了9 nm（0.9 nm）。通过检查几种突变体，我们确定完整的环3对于TonB 2活性不是必需的。结果表明，E.大肠杆菌TonB不是TonB系统在革兰氏阴性细菌物种中的活性所必需的。我们还通过NMR化学位移微扰实验确定了E。coli TonB在体外可与依赖于TonB 2的外膜转运蛋白FatA的TonB盒结合，在体内可替代鳗弧菌铁-鳗毒素转运过程中的TonB 2。出乎意料的是，TonB 2在体外不与FatA TonB-box区域结合，这表明可能需要额外的因子来促进这种相互作用。总的来说，我们的结果表明TonB 2是不同类别的TonB蛋白的代表。
In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.