RESUMO
Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.
Assuntos
Escherichia coli , Sulfurtransferases , Tiossulfatos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Cristalografia por Raios X , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Sulfurtransferases/genética , Sulfurtransferases/metabolismo , Tiossulfatos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.