Characterization of the ligand binding pocket of the virulence regulator Rns, a Member of the AraC/XylS family of transcription factors

Abstract

Diarrheal disease caused by Gram-negative enteric pathogens such as enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella spp., and, Salmonella spp. are a leading cause of morbidity and mortality of children; especially, in low resource nations. While progress has been made in reducing this burden, there remains a need to develop efficacious therapies. Recently, we determined the structure of Rns, a member of the AraC/XylS family that regulates the expression of pili and other virulence factors in ETEC. The structure revealed decanoic acid bound between the N- and C-terminal domains. To test the hypothesis that bound decanoic acid directly inhibits Rns, we used its structure to identify residues that are necessary for ligand binding. Removal of the positive side chains of R75 and H20 rendered Rns insensitive to fatty acid inhibition. Additionally, mutations designed to occlude decanoic acid binding also produced a variant that was fatty acid insensitive. We also observed that this variant is structurally more flexible than wildtype Rns with decanoic acid; suggesting that bound fatty acid contributes to structural rigidity. These studies precisely demonstrate Rns binding pocket residues critical for binding fatty acids and inhibition of DNA binding. This supports our hypothesis that fatty acids must bind in the binding pocket to inhibit AraC regulators. Further work by us and others suggests inhibition of AraC virulence regulators by fatty acids is a common paradigm among many bacterial pathogens. Therefore, understanding the molecular basis of this inhibition lays the groundwork for the development of small molecule therapeutics targeting enteric disease.