Structure, substrate selectivity determinants and membrane interactions of a Glutamate-specific TAXI TRAP binding protein rom Vibrio cholerae.

Tripartite ATP independent periplasmic (TRAP) transporters are widespread in prokaryotes and are responsible for the transport of a variety of different ligands, primarily organic acids. TRAP transporters are secondary active transporters that employ a substrate binding protein to bind and present the substrate to membrane embedded translocation component. TRAP transporters can be divided into two subclasses; DctP-type and TAXI type, which share the same overall architecture and requirement of the SBP for transport, but their SBPs share no similarity. The DctP-type transporters are very well studied and have been shown to transport a range of compounds including dicarboxylates, keto acids, sugar acids. However, the TAXI type transporters are relatively poorly understood, with the range of transportable compounds still to be discovered and selectivity requirements for binding unknown. To address these shortfalls in our understanding, we have structurally and biochemically characterized VC0430 from Vibrio cholerae revealing it to be a monomeric high affinity glutamate binding protein. VC0430 is stereoselective, binding the L-isomer preferentially, and can also bind L-glutamine and L-pyroglutamate, but with low affinity relative to L-glutamate. Structural characterization of ligand bound VC0430 reveals details of the binding site and biophysical characterization of binding site mutant reveal the substrate binding determinants, which differ substantially from the DctP-type TRAPs. Finally, we have analysed in silico the interaction between VC0430 and its cognate membrane component revealing an architecture hitherto unseen. To our knowledge, this is the first transporter in V. cholerae to be identified as specific to glutamate, which plays a key role in osmoadaptation of V. cholerae, making this transporter a potential therapeutic target.