Ligand binding determines proteolytic stability of Vibrio LuxR/HapR quorum sensing transcription factors
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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- p henyl-1-( t hiophen-2-yl s ulfonyl)-1 H - p yrazole) 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.
SIGNIFICANCE
LuxR/HapR proteins were discovered in the 1990s as central regulators of quorum sensing gene expression and later discovered to be conserved in all studied Vibrio species. LuxR/HapR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each LuxR/HapR protein has a predicted ligand binding pocket. However, no ligand has been identified for LuxR/HapR proteins that control their function as regulators. Here, we used LuxR/HapR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation in vivo , the first demonstration of LuxR/HapR function connected to ligand binding for this historical protein family.
