Figure 4. Structural basis for PqsE-induced RhlR activation.

a) Comparison of the closed, inhibited conformation structure of CviR (blue; PDB: 3QP6⁴⁶) bound to the antagonist CL and the open, activated conformation of RhlR (green) bound to the agonist mBTL, PqsE, and DNA. Residue R238 in CviR (blue) and residues K217 and K228 in RhlR (green) are highlighted to display the structurally changes between the closed and active states of LuxR-type proteins. All structure images were created using Pymol. b) E. coli reporter assay expressing RhlR or RhlR variants under the control of the pBAD promoter in the presence of the rhlA promoter fused to luciferase with or without constitutively expressed PqsE. Light levels were normalized to their respective control strain lacking PqsE for each variant. Three biological replicates were used, and each experiment was performed in technical duplicate. Error bars depict standard error of the mean for the biological replicates. P-values were determined by performing a one-way ANOVA with a Dunnett’s multiple comparisons test. Comparisons are only shown relative to the RhlR C157S variant for simplicity. c) Cartoon model for RhlR activation by PqsE. See also Figure S7.