A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.
A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes.
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DOI：
10.3390/ijms23020710
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发表时间：
2022-01-10
影响因子：
5.6
通讯作者：
de Koning HP
中科院分区：
文献类型：
--
作者：
Campagnaro GD;Elati HAA;Balaska S;Martin Abril ME;Natto MJ;Hulpia F;Lee K;Sheiner L;Van Calenbergh S;de Koning HP
刚地弓形虫不能重新合成嘌呤，而是从宿主细胞内的环境中拯救它们，它们需要高亲和力的载体。在这里，我们报告的表达的T。弓形虫平衡核苷转运蛋白，Tg 244440，在核碱基转运蛋白缺失的布氏锥虫菌株中。Tg 244440以相似的亲和力（Km ~1 µM）转运次黄嘌呤和鸟嘌呤，而肌苷和鸟苷的Ki值分别为4.05和3.30 µM。观察到对腺苷、腺嘌呤和嘧啶的低亲和力，将Tg 244440分类为高亲和力氧代嘌呤转运蛋白。嘌呤类似物被用来探测底物转运蛋白结合的相互作用，最终在定量模型显示不同的结合模式，氧代嘌呤碱基，氧代嘌呤核苷，和腺苷。次黄嘌呤和鸟嘌呤通过嘌呤环的质子化N1和N9以及未质子化N3和N7相互作用，而肌苷和鸟苷除了核碱基的N1 H外，主要利用核糖羟基进行结合。相反，腺苷的核糖部分几乎没有对结合做出任何贡献。Tg 244440是第一个在T.并且，据我们所知，第一个嘌呤转运蛋白，采用不同的结合方式的核苷和核碱基。
Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.
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