Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.
Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.
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DOI：
10.1074/jbc.m115.656603
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发表时间：
2015-11-06
期刊：
影响因子：
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通讯作者：
Thomas GH
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文献类型：
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作者：
Fischer M;Hopkins AP;Severi E;Hawkhead J;Bawdon D;Watts AG;Hubbard RE;Thomas GH
背景：流感嗜血杆菌需要依赖底物结合蛋白(SBP)的TRAP转运蛋白来获得唾液酸。结果：SBP中保守的精氨酸残基对于TRAP转运蛋白的高亲和力和羧基专一性是必不可少的。结论：TRAP SBPS中的精氨酸/羧酸相互作用将底物范围限制在含有羧酸的底物上。意义：这项研究揭示了一个关键的双分子相互作用支撑细菌毒力的机制。三聚体ATP非依赖性周质转运蛋白(TRAP)转运蛋白是一种二级转运蛋白，它依赖底物结合蛋白(SBP)来实现单向转运。TRAP SBP的dCTP家族的不同成员具有识别不同范围的有机酸配体的结合位点，但似乎只在配体中保守的精氨酸和羧酸基之间共享共同的静电相互作用。我们利用流感嗜血杆菌毒力相关的SiaPQM TRAP转运蛋白中的唾液酸特异性SBP，研究了这种相互作用的意义。利用体外、体内和结构方法对SIAP进行研究，我们证明了保守的精氨酸(Arg-147)与唾液酸的酸性配体部分的配位对于转运蛋白作为高亲和力清除系统的功能是必不可少的。然而，在高底物浓度下，转运体可以在没有Arg-147的情况下发挥作用，这表明这种双分子相互作用不参与运输周期的进一步阶段。除了高亲和力结合所需的外，我们还证明了Arg-147是陷阱转运蛋白中含羧酸盐底物的强选择性过滤器，方法是通过改造SBP，通过水介导的相互作用识别非羧酸盐底物sialylamide。总之，这些数据提供了捕获转运蛋白的生化和结构支持，这些转运蛋白主要作为含有羧酸的底物的高亲和力转运蛋白发挥作用。
Background: Haemophilus influenzae requires a substrate-binding protein (SBP)-dependent TRAP transporter to acquire sialic acid. Results: A conserved arginine residue in the SBP is essential for the high affinity and carboxylate specificity of the TRAP transporter. Conclusion: The arginine/carboxylate interaction in TRAP SBPs restricts substrate range to carboxylate-containing substrates. Significance: The study reveals the mechanism by which a key bimolecular interaction underpins bacterial virulence. Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.