Locating an extracellular K⁺-dependent interaction site that modulates betaine-binding of the Na⁺-coupled betaine symporter BetP

Abstract

BetP, a trimeric Na þ -coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K þ -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 þ -dependent transport activation and substrate-binding. K þ 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 þ -dependent interactions. A pronounced effect triggered by K þ was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K þ -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 þ -sensing C-terminal domain resulting in a K þ -dependent cooperative betaine-binding.atomic force microscopy | energy landscape | membrane transporter | osmoregulation | secondary substrate-binding site

Impacts of the Osmolality and the Lumenal Ionic Strength on Osmosensory Transporter ProP in Proteoliposomes

“…It led to the concept that BetP responds to the lumenal (and hence the cytoplasmic) K ϩ concentration with an apparent K d of 0.2 to 0.4 M (26). BetP is, therefore, expected to possess a K ϩ -specific regulatory site (27). Analogous work led to the view that OpuA responds to the lumenal and cytoplasmic ionic strength with an I 1/2 of 0.2 M (22).…”

Substrate-induced changes in the structural properties of LacY

“…Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Moreover, when operated in the dynamic mode, dynamic single-molecule force spectroscopy (DFS) localizes and quantifies the kinetic, energetic,…”

Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor

“…Measuring the kinetic response of the individual structural elements by dynamic SMFS (DFS) allows quantification of their unfolding free energy, kinetic stability, conformational variability, and mechanical flexibility (33). Both SMFS and DFS have been applied to characterize these parameters of various membrane proteins and follow how they change on changing their functional state or physiological environment (34)(35)(36)(37)(38)(39).…”