Lefebvre Solène N, Nijland Mark, Maslov Ivan, Slotboom Dirk J
Faculty of Science and Engineering, Groningen, Biomolecular Sciences and Biotechnology, Membrane Enzymology Group, University of Groningen, Groningen, The Netherlands.
Nat Commun. 2025 May 13;16(1):4448. doi: 10.1038/s41467-025-59674-6.
Energy-Coupling Factor (ECF) transporters are ATP-binding cassette (ABC) transporters essential for uptake of vitamins and cofactors in prokaryotes. They have been linked to pathogen virulence and are potential targets for antimicrobials. ECF transporters have been proposed to use a unique transport mechanism where a substrate-translocating subunit (S-component) dynamically associates with and dissociates from an ATP-hydrolyzing motor (ECF module). This model is contentious, because it is based largely on experimental conditions without compartments or continuous bilayers. Here, we used single-molecule spectroscopy to investigate the conformational dynamics of the vitamin B12 transporter ECF-CbrT in membranes under vectorial transport conditions. We observed ATP hydrolysis-dependent dissociation of the S-component CbrT from, and re-association with the ECF module, in absence and presence of vitamin B12 consistent with futile ATP hydrolysis activity. The single-molecule spectroscopy experiments suggest that S-component expulsion from and re-association with the ECF module are an integral part of the translocation mechanism.
能量偶联因子（ECF）转运蛋白是ATP结合盒（ABC）转运蛋白，对于原核生物摄取维生素和辅因子至关重要。它们与病原体毒力有关，是抗菌药物的潜在靶点。有人提出ECF转运蛋白使用一种独特的转运机制，其中底物转运亚基（S-组分）与ATP水解马达（ECF模块）动态结合和解离。该模型存在争议，因为它主要基于没有分隔或连续双层的实验条件。在这里，我们使用单分子光谱来研究在矢量运输条件下膜中维生素B12转运蛋白ECF-CbrT的构象动力学。我们观察到在不存在和存在维生素B12的情况下，S-组分CbrT与ECF模块的ATP水解依赖性解离和重新结合，这与无效的ATP水解活性一致。单分子光谱实验表明，S-组分从ECF模块排出和重新结合是转运机制的一个组成部分。
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