Bacteria evolve mechanisms to compete for limited resources and survive in new niches. Here we study the mechanism of isethionate import from the sulfate-reducing bacterium Oleidesulfovibrio alaskensis. The catabolism of isethionate by Desulfovibrio species has been implicated in human disease, due to hydrogen sulfide production, and has potential for industrial applications. O. alaskensis employs a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM) to import isethionate, which relies on the substrate-binding protein (OaIseP) to scavenge isethionate and deliver it to the membrane transporter component (OaIseQM) for import into the cell. We determined the binding affinity of isethionate to OaIseP by isothermal titration calorimetry (ITC), KD = 0.95 µM (68% CI = 0.6-1.4 µM), which is weaker compared to other TRAP substrate-binding proteins. The X-ray crystal structures of OaIseP in the ligand-free and isethionate-bound forms were obtained and showed that in the presence of isethionate, OaIseP adopts a closed conformation whereby two domains of the protein fold over the substrate. We serendipitously discovered two crystal forms with sulfonate-containing buffers (HEPES and MES) bound in the isethionate-binding site. However, these do not evoke domain closure, presumably because of the larger ligand size. Together, our data elucidate the molecular details of how a TRAP substrate-binding protein binds a sulfonate-containing substrate, rather than a typical carboxylate-containing substrate. These results may inform future antibiotic development to target TRAP transporters and provide insights into protein engineering of TRAP transporter substrate-binding proteins.

中文翻译：

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关于 TRAP 底物结合蛋白的功能：羟乙基磺酸盐特异性结合蛋白 IseP。


细菌进化出机制来争夺有限的资源并在新的生态位中生存。在这里，我们研究了从硫酸盐还原细菌 Oleidesulfovibrio alaskensis 输入羟乙磺酸的机制。由于硫化氢的产生，Desulfovibrio 物种对羟乙基磺酸盐的分解代谢与人类疾病有关，并具有工业应用的潜力。O. alaskensis 采用三方 ATP 非依赖性周质 （TRAP） 转运蛋白 （OaIsePQM） 导入羟乙基磺酸盐，它依靠底物结合蛋白 （OaIseP） 清除羟乙基磺酸盐并将其输送到膜转运蛋白成分 （OaIseQM） 以输入细胞。我们通过等温滴定量热法 （ITC） 确定了羟乙基磺酸盐与 OaIseP 的结合亲和力，KD = 0.95 μM （68% CI = 0.6-1.4 μM），与其他 TRAP 底物结合蛋白相比较弱。获得了无配体和异乙基磺酸结合形式的 OaIseP 的 X 射线晶体结构，结果表明在异羟基异丙氨酸存在下，OaIseP 采用封闭构象，即蛋白质的两个结构域在底物上折叠。我们偶然发现了两种晶体形式，其中含磺酸盐的缓冲液 （HEPES 和 MES） 结合在乙乙基磺酸盐结合位点。然而，这些不会引起结构域闭合，可能是因为配体大小较大。总之，我们的数据阐明了 TRAP 底物结合蛋白如何结合含磺酸盐的底物，而不是典型的含羧酸盐的底物的分子细节。这些结果可能为未来靶向 TRAP 转运蛋白的抗生素开发提供信息，并为 TRAP 转运蛋白底物结合蛋白的蛋白质工程提供见解。