Structural and Functional Characterisation of a Novel TAXI TRAP Binding Protein from Bordetella pertussis

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary active transporters found within prokaryotes that are dependent on a substrate binding protein (SBP) and two uneven transmembrane components for the translocation of substrates across the membrane. The presence of an SBP allows for increased affinity and specificity of substrates thus resulting in a highly efficient transporter that is able to gather nutrients in the most depleted and competitive of environments. The TAXI (TRAP associated immunogenic) subclass of TRAP transporters has been neglected from literature, however their SBPs have been shown to follow the same architecture as the better characterised DctP-type TRAPs. This consists of a 300-350 amino acid monomeric bilobular protein composed of 2 α/β globular regions connected via a flexible hinge.

Within this study, we highlight the discovery of a TAXI TRAP SBP from Bordetella Pertussis with an unusual residue length and architecture. This SBP is 503 residues in length and is composed of an α/β globular N-terminal domain, an interdomain helical region and a domain resembling a TRAP SBP. Through (1) size exclusion chromatography (2) computational modelling and (3) the introduction of site-specific cysteine mutations we uncover this TAXI TRAP SBP to be a stable dimer with the dimerisation point being within the interdomain helical region. Following this, differential scanning fluorimetry and intrinsic tyrosine fluorescence reveal this SBP to bind to L-glutamate with high affinity. We also uncover five other potential glutamate binding dimeric TAXI SBPs, highlighting a possible novel family of dimeric SBP- dependent secondary active transporters.