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首页> 外文期刊>Journal of Molecular Biology >The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline.
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The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline.

机译：枯草芽孢杆菌底物结合蛋白OpuBC与胆碱复合的晶体结构。

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Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K(d) value of about 30 muM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 A and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.
机译：枯草芽孢杆菌可从前体胆碱的外源供应中合成相容的溶质甘氨酸甜菜碱作为渗透保护剂。胆碱的导入是由两种渗透诱导的ABC转运系统介导的：OpuB和OpuC。 OpuC催化各种渗透保护剂的导入，而OpuB对胆碱具有高度特异性。 OpuBC是OpuB转运蛋白的底物结合蛋白，我们已经通过固有色氨酸荧光分析了OpuBC /胆碱复合物的亲和力，并确定了约30μM的K（d）值。 OpuBC /胆碱复合物的X射线晶体结构以1.6 A的分辨率解析，并显示出II类底物结合蛋白的典型折叠。胆碱的带正电荷的三甲基铵头部基团楔入由四个酪氨酸残基形成的芳族笼中，并通过阳离子-pi相互作用结合。胆碱的羟基从该芳香族笼中伸出，并与残基Gln19单键相互作用。该残基被Ala取代会使胆碱结合亲和力降低约15倍。水网络将胆碱稳定在其底物结合位点内，并促进OpuBC蛋白的两个叶之间的间接相互作用。通过对OpuBC中选定残基进行定点诱变来破坏这种复杂的水网络，或者会大大降低胆碱结合亲和力（在18倍至25倍之间），或者取消配体结合。 OpuBC /胆碱复合物的晶体结构为体内观察到的OpuB ABC导入物的胆碱特异性提供了合理的理由，并解释了其无法催化甘氨酸甜菜碱导入渗透压枯草芽孢杆菌细胞中。

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