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Abstract

The trimeric Na⁺-coupled betaine symporter BetP from Corynebactrium glutamicum adjusts transport activity according to the external osmolality. BetP senses the increasing internal K⁺ concentration, which is an immediate consequence of osmotic upshift in C. glutamicum. It is assumed that BetP specifically binds potassium to yet unidentified binding sites, thereby inducing conformational changes resulting in activation. Atomic structures of BetP were obtained in the absence of potassium allowing only a speculative glimpse on a putative mechanism of K⁺-induced transport activation. The structural data suggest that activation in BetP is crucially linked to its trimeric state involving an interaction network between several arginines and glutamates and aspartates. Here, we describe the effect of K⁺-induced activation on the specific ionic interaction sites in terminal domains and loops and on the protomer–protomer interactions within the trimer studied by ATR-FTIR spectroscopy. We suggest that arginine and aspartate and/or glutamate residues at the trimeric interface rearrange upon K⁺-induced activation, although they remain assembled in an interaction network. Our data propose a two-step mechanism comprising first a change in solvent exposure of charged residues and second a modification of their interaction sites in a partner-switching manner. FTIR reveals a higher α-helical content than expected from the X-ray structures that we attribute to the structurally unresolved N-terminal domain modulating regulation. In situ ¹H/²H exchange studies point toward an altered exposure of backbone regions to buffer solution upon activation, most likely due to conformational changes in both terminal domains, which further affects ionic interactions within the trimer.