Kim, T.-Y.; Ahn, W.-C.; Kim, J.-A.; Lee, K.-W.; Kim, S.; Park, K.-H.; Woo, E.-J.; Kim, K.-S.

In Vibrio vulnificus, the quorum-sensing signal cyclo-(L-phenylalanine-L-proline) (cFP) binds the membrane receptor ToxRS to activate genes linked to oxidative-stress resistance and virulence. ToxR is a transmembrane transcription factor that pairs with ToxS to sense periplasmic signals, yet how V. vulnificus ToxRS recognizes cFP has remained undefined. AI-guided structure prediction revealed preferential ToxR/S heterodimer formation and a fold conserved with the V. cholerae crystal structure. Molecular docking placed cFP in a hydrophobic pocket at the ToxR-ToxS interface, where Phe279 stacked with its phenyl ring and Arg277 hydrogen-bonded its carbonyl oxygen. Introducing R277L and F279A substitutions lowered basal leuO expression and abolished cFP-dependent induction in a lacZ fusion assay. ChIP showed that cFP enhanced wild-type ToxR binding to the leuO promoter, whereas the mutant bound weakly and did not respond. Thus cFP bridges ToxR and ToxS to stabilize the heterodimer, facilitating ToxR recruitment to promoters and transcriptional activation in V. vulnificus.