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Abstract

BetP, a trimeric Na⁺-coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K⁺-concentration. This transport regulation of BetP depends on a sophisticated interaction network. Using single-molecule force spectroscopy we structurally localize and quantify these interactions changing on K⁺-dependent transport activation and substrate-binding. K⁺ significantly strengthened all interactions, modulated lifetimes of functionally important structural regions, and increased the mechanical rigidity of the symporter. Substrate-binding could modulate, but not establish most of these K⁺-dependent interactions. A pronounced effect triggered by K⁺ was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K⁺-concentrations akin to those BetP encounters during hyperosmotic stress trigger the formation of a periplasmic second betaine-binding (S2) site, which was found to be at a similar position reported previously for the BetP homologue CaiT. In BetP, the presence of the S2 site strengthened the interaction between EH2, transmembrane α-helix 12 and the K⁺-sensing C-terminal domain resulting in a K⁺-dependent cooperative betaine-binding.