Substrate Bound Outward-Open State of the Symporter BetP: Insights Into Sodium and Substrate Binding and Coupling

Abstract

BetP is a Na+-coupled symporter that is able to accumulate betaine in molar amounts during hyperosmotic stress by coupling substrate transport to the Na+ electrochemical potential. BetP shares the highly conserved fold of two-inverted structural repeats with other sequence unrelated secondary transporters, e.g., LeuT, a bacterial homolog of the medically important neurotransmitter transporter family. Recently, we obtained atomic structures from BetP in distinct conformational states, elucidating parts of the alternating access mechanism of BetP, and suggesting a common mechanistic principle in LeuT-like fold transporters. However, as the substrate-bound outward-open state of BetP was still lacking from the collection of conformational states, the fundamental question about the first coupling step, i.e., the sequential binding of substrate and sodium ions, remained unanswered. Here, we report on a structure of a Na+-coupled choline-transporting BetP mutant (G153D) in the substrate-bound outward-open state in complex with arseno-choline, which allows us to complete the molecular description of the alternating-access cycle of BetP. Our structural results confirm substrate binding by arsenic anomalous scattering, and in combination with molecular dynamics simulations, reveal key features of the formation of the central substrate-binding site and the two sodium ion-binding sites. The sequential conformational changes from outward-open to closed state provide us with further insights into the nature of the coupled binding of trimethylammonium substrates and the two sodium ions to BetP and consequently their impact on the state transitions in the alternating-access cycle.