Fig. 3: AP2-G.L DNA binding is disrupted.
a Motif search (https://www.genome.jp/tools/motif/) computational prediction of the amino acids (aa) contributing to the AP2 domain (red text) in AP2-G with the indicated amino acid at position 2163 (green box) and the independent expectation value (i-Evalue). b–f AlphaFold30,31 3D modeling predicts the structure of the domain (aa 2098-2245, green) that includes the AP2 domain (aa 2162-2210, purple) (c) with high confidence in the context of the full length wt AP2-G.V (d). The predicted local distance difference test (pLDDT) was used to evaluate confidence (b) and color code the structure, very high (dark blue >90%), high (cyan: 70-90%), low (yellow: 50-70%), very low (orange: 50%) (d, f). The dashed gray box indicates the amino acids in the recombinant AP2-G used in the electrophoretic mobility shift assay (EMSA) (f). e MutationExplorer58 analysis predicted local energy changes in full length AP2-G.L compared to wt AP2-G.V. The red bubble indicates the mutation sphere and residues that are stabilized or destabilized, respectively. f The AlphaFold prediction for recombinant mutant AP2-G.L (aa 2150-2220) used in the EMSA color coded based on pLDDT model confidence. The pink square indicates the mutant L2163 and the circles correspond to residues, I2154 and R2201 that are predicted to be destabilized using MutationExplorer. g EMSA analysis using a light shift EMSA assay kit with biotinylated probe GTGTACAC and recombinant proteins with either a wild type or mutated AP2 domain, P. falciparum recombinant AP2-G AP2 domain, aa 2150-2220 (Pf-WT or Pf-V2163L) or P. berghei wild type AP2-G AP2 domain (Pb-WT)63. Shifted and unshifted bands are indicated by respective arrow. The EMSA experiment was performed once at 2 different recombinant protein concentrations. The uncropped gel is included in the Source Data file.
