Conformational transition induced in the aspartate:alanine antiporter by L-Ala binding.

Autor: Suzuki S; Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan., Chiba F; Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan., Kimura T; Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan., Kon N; Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan., Nanatani K; Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan. kei.nanatani.a7@tohoku.ac.jp.; Structural Biology Group, Advanced Research Center for Innovations in Next-Generation Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8573, Japan. kei.nanatani.a7@tohoku.ac.jp., Abe K; Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan. keietsu.abe.b5@tohoku.ac.jp.; Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8579, Japan. keietsu.abe.b5@tohoku.ac.jp.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2022 Sep 23; Vol. 12 (1), pp. 15871. Date of Electronic Publication: 2022 Sep 23.
DOI: 10.1038/s41598-022-19974-z
Abstrakt: An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate 1- :alanine 0 exchange reaction. Our previous kinetic analyses of transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of L-aspartate (-Asp) and L-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of L-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of L-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of L-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (L-Ala or L-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of L-Ala differed from that in the presence of L-Asp. From these results, we concluded that L-Ala binding yields a conformation different from the apo or L-Asp binding conformations.
(© 2022. The Author(s).)
Databáze: MEDLINE
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