Fig. 4: Mutations increase selectivity of MAX by enhancing cognate or weakening mutant DNA binding.
a Cartoon depicting additive and epistatic energetic impacts for double mutants (top); pairwise comparisons between measured ΔΔG (median ± SEM) relative to WT for all MAX mutants interacting with low-affinity 5′-C CACGCG A−3′ versus consensus 5′-C CACGTG A-3′. Light gray markers indicate mutations unresolvable from background for ≥1 DNA sequence; black markers indicate known crystallographic contacts to mutated nucleotide bases; red marker edges indicate non-additive binding; dashed black line indicates linear regression; red dashed line indicates 1:1 identity line. b Mutants with epistatic energetic impacts projected onto MAX structure (1HLO). c Schematic illustrating calculation of selectivity scores from double mutant cycles. d Selectivity scores vs. ΔΔGCACGTG (median ± SEM) for all MAX mutations. Light gray markers indicate mutations unresolvable from background for ≥1 DNA sequence; dashed gray line indicates thresholds for classifying mutations. e–h Relative affinities (left) and median Kd ± SEM (right) across all E-box variants for WT and selected MAX mutants, with location of mutation highlighted on structure (1HLO and 5EYO). Replicate counts for all Kd measurements are contained in Supplementary Data 1. i Cartoon depicting double mutant cycle analysis to probe for energetic coupling between selective TF mutations (left); ΔΔG (median ± SEM) for 2 combinations of selective mutations (right), with expected additive and measured ΔΔGs compared via independent t-test (***p < 0.001; **p < 0.01; *p < 0.05). Source data are provided as a Source Data file.
