Lock and Load: Key Role of the Unique Closed State in Transport Regulation of the Sodium-Coupled Betaine Transporter BetP

The Na+-coupled betaine symporter BetP is regulated by osmotic stress sensing the increasing internal K+ concentration as a direct consequence of hypertonicity by its positively charged C-terminal domain. Recently, we demonstrated that BetP binds K+ cooperatively to its cytoplasmic side between different C-terminal domains within the BetP trimer, while the monomer is not regulated. K+ binding strengthens the helical fold of the C-terminal domain, and together with negatively charged lipids at the trimer center orients it towards the counterclockwise adjacent protomer. The extended C-terminal conformation is assisted by an additional interaction with the N-terminal domain and most crucially with lipids. A recent crystal structure show BetP in a closed state with the C-terminal domain bending back towards its own protomer to interact mainly with lipid head groups at the membrane surface. Thereby, an intratrimeric interaction is inhibited, which allows us to assign the bend-back conformation of the C-terminal domain as the inactive one and the extended conformation as active. A surprising consequence of the inactive C-terminal conformation is the uncoupling of the two sodium sites. Although having all substrates bound BetP cannot any longer isomerize to the inward-facing state because the C-terminal domain together with lipids lock the intracellular gate preventing the opening of the S1 site. Subsequently, all BetP protomers within a trimer remain fully substrate loaded in a closed state with additional betaine molecules sequestered and stored in the second periplasmic (S2) binding site. We propose a trimeric switch model, in which osmotic stress induces a stepwise activation of one protomer after the other by consecutive K+ binding and straightening of the C-terminal helix.