{"title":"MATE 转运体的亲和性底物/H+-反转运作用","authors":"Koh Takeuchi, Takumi Ueda, Misaki Imai, Miwa Fujisaki, Mie Shimura, Yuji Tokunaga, Yutaka Kofuku, Ichio Shimada","doi":"10.1016/j.str.2024.05.004","DOIUrl":null,"url":null,"abstract":"<p>Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na<sup>+</sup>/H<sup>+</sup> influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of <em>Pyrococcus furiosus</em> MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H<sup>+</sup> antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H<sup>+</sup>-coupled substrate excretion by the transporter.</p>","PeriodicalId":22168,"journal":{"name":"Structure","volume":null,"pages":null},"PeriodicalIF":4.4000,"publicationDate":"2024-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Affinity-directed substrate/H+-antiport by a MATE transporter\",\"authors\":\"Koh Takeuchi, Takumi Ueda, Misaki Imai, Miwa Fujisaki, Mie Shimura, Yuji Tokunaga, Yutaka Kofuku, Ichio Shimada\",\"doi\":\"10.1016/j.str.2024.05.004\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na<sup>+</sup>/H<sup>+</sup> influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of <em>Pyrococcus furiosus</em> MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H<sup>+</sup> antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H<sup>+</sup>-coupled substrate excretion by the transporter.</p>\",\"PeriodicalId\":22168,\"journal\":{\"name\":\"Structure\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.4000,\"publicationDate\":\"2024-05-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Structure\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1016/j.str.2024.05.004\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Structure","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/j.str.2024.05.004","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
多药和毒素挤出(MATE)家族转运体排出有毒化合物与 Na+/H+ 流入有关。虽然已有 MATE 转运体的结构,但底物输出与离子流入耦合的机制仍然未知。为了解决这个问题,我们利用溶液核磁共振(NMR)对暴怒双球菌 MATE(PfMATE)进行了结构分析。核磁共振分析以及对所有未暴露的酸性残基进行的彻底置换证实,PfMATE 在由 Glu163 质子化决定的内向型(IF)和外向型(OF)构象之间处于平衡状态。重要的是,我们发现只有 IF 构象对底物的识别具有中等微米的亲和力。相比之下,OF 构象只表现出微弱的 mM 底物亲和力,适合将底物释放到胞外侧。这些结果表明,PfMATE 是一种亲和力导向的 H+ 反转运体,底物在平衡状态下选择性地与质子化的 IF 构象结合,随后的质子释放从机理上确保转运体排出 H+ 耦合的底物。
Affinity-directed substrate/H+-antiport by a MATE transporter
Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.
期刊介绍:
Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome.
In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.