Gallic Acid-induced Tetramerisation of the Pleiotropic Virulence Factor SuhB May Alter Its Regulatory Role in P....

Nus-factor SuhB protein from Pseudomonas aeruginosa (PaSuhB) is a pleiotropic regulator of multiple genes associated with the virulence of the pathogen. We have solved the crystal structures of PaSuhB in its apo and substrate-bound (D-myo-Inositol-1-phosphate) forms. 3D-pharmacophore modelling of the PaSuhB-bound substrate identified gallic acid (GDE) as a potential inhibitor of the protein. In-vitro enzyme-kinetics studies indicate that GDE is a non-competitive inhibitor of PaSuhB. The high-resolution crystal structure of the GDE-PaSuhB complex uncovers the existence of a cryptic ligand-binding site, located at the distal site of the protein's active site pocket. This cryptic site was identified at the dimerisation interface of the protein, in proximity of the α4 helix. Detailed structural analysis reveals that GDE, bound at the cryptic site of PaSuhB, induces structural distortion of the α4 helix and thereby promotes an asymmetric tetramer formation of dimeric PaSuhB. 'In-solution' dynamic light scattering and size-exclusion chromatography experiments further fortify the GDE-mediated tetramerisation of PaSuhB. The asymmetric tetramer formation of PaSuhB shields the conserved NusA-AR2 domain binding site of the protein and may impact its extragenic suppressor activity. In vitro, GDE at non-lethal concentrations induced the 'cold-sensitive' growth phenomenon in P. aeruginosa and E. coli, a characteristic feature of the suhB deletion mutant. GDE also inhibits the swimming motility of the bacteria. Altogether, our results demonstrated a novel cryptic ligand binding site proximal to the dimerisation interface of pleiotropic virulence regulator SuhB from P. aeruginosa, which can be exploited further to design specific therapeutic leads against this dreaded pathogen.