Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding pathways

Understanding how proteins discriminate between preferred and non-preferred ligands (‘selectivity’) is essential for predicting biological function and a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study how variants of the promiscuous transcription factor (TF) MAX (H. sapiens) alter DNA specificity and selectivity, yielding >1700 K_ds and >500 rate constants in complex with multiple DNA sequences. Twenty-two of the 240 assayed MAX point mutations enhance selectivity, yet none of these mutations occur at residues that contact nucleotides in published structures. By applying thermodynamic and kinetic models to these results and previous observations for the highly similar yet far more selective TF Pho4 (S. cerevisiae), we find that these mutations enhance selectivity by altering partitioning between or affinity within conformations with different intrinsic selectivity, providing a mechanistic basis for allosteric modulation of ligand selectivity. These results highlight the importance of conformational heterogeneity in determining sequence selectivity and can guide future efforts to engineer selective proteins.