Fig. 3: Structure-guided mutations in FasR leading to permanent DNA-binding.
a Selected mutations to occlude the effector-binding tunnel are illustrated. The crystal structure of FasRΔ33-C20-CoA (left panel) is compared to a model of the triple mutant FasRLVL (right panel). Val163 is actually behind the tunnel (red arrow). The substitutions by bulky phenylalanines anticipate steric hindrance of the acyl chain (shown as sticks coloured by element). b Electrophoretic mobility shift assay was performed by incubating the 32P-labelled 398 bp Pfas promoter region with wild-type FasR and mutant FasR proteins (FasRLVL and FasRL106F) in the absence or in the presence of C20-CoA and C16-CoA. Protein–DNA complexes were separated by electrophoresis on a 6% polyacrylamide gel. The assay was performed in duplicate, producing similar results. Source data are provided as a Source Data file. c Selected mutations to uncouple allosteric signalling from the ligand-binding pocket to the DNA-binding domain. The bottom mouth of the effector-binding tunnel is located towards the centre of the molecule, revealing the tip of C20 (in ball and stick representation, coloured by atom). A few side chains are labelled and shown as orange sticks, chosen among residues bordering the tunnel opening. Two bulky residues (Leu98 on helix α4 and Phe123 on α5) that were substituted by alanines, are labelled with bold fonts. d Gel shift assays of the wild-type and selected FasR mutants. The DNA probe that corresponds to the 398 bp Pfas promoter region (PfasMT) was 32P-labelled and incubated either with FasR wild-type, FasRL98A or FasRF123A, in the absence or in the presence of the indicated concentrations of C16-CoA and C20-CoA. The assays were performed in duplicate, producing similar results. Source Data are provided as a Source data file.
