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Abstract

The glycine betaine/sodium symporter BetP responds to changes in external osmolality by regulation of its transport activity. A recent X-ray structure of BetP confirms that it is a homotrimer and in this structure each protomer adopts an identical conformation, in which the pathway is occluded from both sides. Despite the availability of a wealth of experimental data for BetP, the structures of the alternate states (e.g., open to the outside of the cell), molecular mechanisms of substrate and Na⁺ binding and transport, as well as the functional implications of the trimeric state remain poorly understood. To address these questions, we carried out computational studies using a range of techniques to derive hypotheses that were then tested experimentally. First, to identify structural features of the alternate states, we developed a procedure for flexible fitting of the X-ray structure of BetP into a lower-resolution cryo-EM map of BetP in a more native lipid environment, in which the three protomers have different conformations. These results suggest that: (i) the protomers adopt distinct conformational states relevant to the transport cycle; and (ii) there is conformational coupling between the protomers. Second, we performed all-atom molecular dynamics simulations and in silico alanine scanning of BetP trimers in order to identify interface residues crucial for maintaining the trimeric state. Mutations of these residues to alanine were introduced experimentally revealing that the isolated monomers are functional, and that the trimeric state is important for the regulation and higher activity of the protein. Finally, using molecular modeling and biochemical experiments we identified two Na⁺ binding sites in BetP that could not be resolved in the 3.35 Å resolution X-ray structure.