Figure 8.

For each variant, the binding free energy changes (ΔΔG_binding) in kcal/mol were computed using averaged values across CDKL5 binding partners, and the ΔΔG_Bmax threshold was subsequently determined. (A) Multi-panel bar charts of absolute ΔΔG_binding for CDKL5-target protein complexes at their consensus phosphosite motifs. (A1–A4) Binding free energy changes (ΔΔG_binding) due to benign variants. (A5–A8) Changes in ΔΔG_binding due to pathogenic variants for (A1,A5) CDKL5-AMPH1 (motif 290–294), (A2,A6) CDKL5-GATAD2A (motif 97–101), (A3,A7) CDKL5-SOX9 (motif 197–202), and (A4,A8) CDKL5-ZNF219 (motif 111–115). In each panel, opaque colored bars show the mean |ΔΔG_binding| across methods for each variant. Variant labels include this complex average across methods in parentheses (e.g., “I3F (0.58)”). Legends above the panels represent the type of bars (methods and average). (B) Sorted bar plot of ΔΔG_Bmax: the maximum complex average |ΔΔG_binding| across the four CDKL5-target protein motifs for all thirteen variants (four benign and nine pathogenic). Blue bars denote benign; orange bars denote pathogenic. Numerical ΔΔG_Bmax values are labeled above each bar. The dashed gray line indicates the classification cutoff (0.88 kcal/mol), defined as the midpoint between the highest benign ΔΔG_Bmax (0.82 kcal/mol) and the lowest pathogenic ΔΔG_Bmax (0.95 kcal/mol).