Structural insights into the specificity of a bacterial pyridoxal 5’-phosphate periplasmic binding protein

Vitamin B6 and its vitamers are essential cofactors in bacteria, with about 1.5% of bacterial and archaeal genes encoding pyridoxal 5′-phosphate (PLP)-dependent enzymes. While many bacteria rely on salvage pathways and transport systems due to B6 auxotrophy, even those capable of de-novo synthesis often retain uptake mechanisms, likely to reduce metabolic costs. Despite their importance, few B6 transporters have been structurally characterized. The crystallographic structure of the PLP-binding protein P5PA from Actinobacillus pleuropneumoniae, a swine pathogen, has provided key insights into PLP recognition, particularly when compared to the structurally similar sugar-binding protein AfuA. This study explored the molecular basis of ligand recognition by combining cross-docking of heterologous complexes (P5PA- Glucose-6-phosphate (G6P) and AfuA-PLP) with molecular dynamics simulations of all complexes, each run for 500 ns in triplicate. Simulations revealed increased ligand instability when bound to non-native proteins, supporting the idea that each protein exhibits high specificity for its physiological ligand. Interaction energy analysis confirmed stronger binding in native complexes. In P5PA, Q267 residue forms a key electrostatic interaction with PLP that is lost when G6P is bound. Conversely, in AfuA, D206 residue stabilizes G6P but does not interact with PLP. Notably, this position corresponds to a non-polar residue (A204) in P5PA, further highlighting their functional divergence. Conformational clustering revealed that the presence of different ligands induces distinct structural rearrangements in each protein. These findings deepen our understanding of PLP recognition and may aid in identifying related transporters or designing antibacterial agents targeting vitamin B6 uptake systems.