Figure 5: Role of the key residues in the HTH motif region in GrlR–GrlAΔ interactions.
From: Structure of GrlR–GrlA complex that prevents GrlA activation of virulence genes
(a) Pull-down assay to show the binding of GrlAΔ or mutant variants with GrlR. MBP-GrlAΔ proteins were bound to amylose beads, incubated with 6His-GrlR overnight and washed. The eluted proteins were separated by 12.5% SDS–PAGE and the gels stained with Coomassie brilliant blue. The GrlA derivatives employed are indicated above the lanes: MBP-GrlAΔ (R65A K66A), MBP-GrlAΔ (R64A R65A), MBP-GrlAΔ (R53A R54A), WT MBP-GrlAΔ and MBP. (b) Pull-down assay to show the binding of GrlAΔ with GrlR or the GrlR mutants. The GrlR mutants employed are indicated above the lanes: GrlR D17A, GrlR N66A, GrlR S18A, GrlR D35A GrlR E60A and WT GrlR. The input washes and final elution of the representative mutants are shown in Supplementary Fig. S4a. The additional bands seen on the SDS–PAGE (a) were identified as MBP using peptide mass fingerprint analysis (Supplementary Fig. S4b). Western blot analysis of the pull-down assay (a,b) was carried out using anti-MBP and anti-His antibodies (Supplementary Fig. S5a,d). The pull-down results were confirmed in a co-expression, co-purification pETDuet-grlR-grlAΔ system and analysed on a 12.5% native gel (Supplementary Figs S6a,b, S7 and S8).
