Ligand binding determines proteolytic stability ofVibrioLuxR/HapR quorum sensing transcription factors

Abstract

In Vibrio species, quorum sensing signaling culminates in the production of a TetR-type master transcription factor collectively called the LuxR/HapR family, which regulates genes required for colonization and infection of host organisms. These proteins possess a solvent accessible putative ligand binding pocket. However, a native ligand has not been identified, and the role of ligand binding in LuxR/HapR function in Vibrionaceae is unknown. To probe the role of the ligand binding pocket, we utilize the small molecule thiophenesulfonamide inhibitor PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole) that we previously showed targets LuxR/HapR proteins. Amino acid conservation in the ligand binding pocket determines the specificity and efficacy of PTSP inhibition across Vibrio species. Here, we used structure-function analyses to identify PTSP-interacting residues in the ligand binding pocket of SmcR - the Vibrio vulnificus LuxR/HapR homolog - that are required for PTSP inhibition of SmcR activity in vivo. Forward genetic screening combined with X-ray crystallography structural determination of SmcR bound to PTSP identified substitutions at eight residues that were sufficient to reduce or eliminate PTSP-mediated SmcR inhibition. Small-angle X-ray scattering and computational modeling determined that PTSP drives allosteric unfolding at the N-terminal DNA binding domain. We discovered that SmcR is degraded by the ClpAP protease in the presence of PTSP in vivo; substitution of key PTSP-interacting residues stabilized or increased SmcR levels in the cell. This mechanism of inhibition is observed for all thiophenesulfonamide compounds tested and against other Vibrio species. We conclude that thiophenesulfonamides specifically bind in the ligand binding pocket of LuxR/HapR proteins, promoting protein degradation and thereby suppressing downstream gene expression, implicating ligand binding as a mediator of LuxR/HapR protein stability and function to govern virulence gene expression in Vibrio pathogens.