Characterization of a TAXI-TRAP Substrate-Binding Protein from Vibrio cholerae

TAXI-TRAP transporters represent a class of secondary substrate-binding protein (SBP)- dependent systems that are utilized by certain bacteria for the high affinity uptake and transport of substrates across the membrane. A limited number of studies have provided evidence of the substrate range of some TAXI-TRAP SBPs, but a full biochemical characterization of one of these essential components of a TAXI-TRAP system is still absent from literature.

In this study, we characterize a SBP, VC0430, from a TAXI-TRAP system that was identified in the facultative pathogen, Vibrio cholerae. Analysis of genes that are within the same genetic neighbourhood as vc0430 suggested a function of the protein in the transport of carboxylic

acids across the membrane. We capitalized on this clue by generating an accurate homology model of VC0430 and then mapped its binding site against the only TAXI-TRAP SBP crystal structure that is available in literature. Along with the insight provided by a multiple sequence alignment between the two proteins, this binding site superimposition enabled us to identify conserved residues in VC0430 that are involved in the binding of glutamate and glutamine.

We proved this predicted substrate range to be correct through the use of thermal shift assays, whereby glutamate and glutamine were the only substrates to increase the thermal stability of the protein.

Lastly, having proven that VC0430 is a glutamate/glutamine binding protein, we speculated the significance of these two substrates to Vibrio cholerae and suggested the part that VC0430 could play in the survival of the bacterium. As glutamate, and hence glutamine as its metabolic precursor, has an important role in the osmoadaptation of Vibrio cholerae to both the estuarine and human intestinal environment, we identify VC0430 as a potential drug target to combat disease caused by this pathogen. We also provide experimental methods that can help further characterize the TAXI-TRAP family in future studies.