title: Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP.
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.
authors: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, 4058 Basel, Switzerland.
authors: Lin
authors: L
authors: Ge
authors: Camilo
authors: C
authors: Perez
authors: Izabela
authors: I
authors: Waclawska
authors: Christine
authors: C
authors: Ziegler
authors: Daniel J
authors: DJ
authors: Muller
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D005453:Fluorescence; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D008958:Models, Molecular; D011188:Potassium / Q000378:metabolism*; D027981:Symporters; D014882:Water-Electrolyte Balance / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D002352:Carrier Proteins; D027981:Symporters; D001622:Betaine; D011188:Potassium