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Abstract

BetP is an Na⁺-coupled betaine-specific transporter of the betaine–choline–carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT-fold) that BetP shares with other sequence-unrelated Na⁺-coupled symporters. Numerous structures of LeuT-fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and co-transported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a single-point mutation—glycine to aspartate—into an H⁺-coupled choline-specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP-G153D demonstrates a new inward-facing open conformation for BetP. Choline binding to a location close to the second, low-affinity sodium-binding site (Na2) of LeuT-fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cation-binding site in BetP, playing a key role in a proposed molecular mechanism of Na⁺ and H⁺ coupling in BCC transporters.