Enhancing Binding Affinity of an Intrinsically Disordered Protein by α-Methylation of Key Amino Acid Residues

Intrinsically disordered proteins (IDPs), which undergo folding upon binding to their targets, are critical players in protein interaction networks. Here we demonstrate that incorporation of non-canonical alpha-methylated amino acids into the unstructured activation domain of the transcriptional coactivator ACTR can stabilize helical conformations and strengthen binding interactions with the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP). A combinatorial alpha-methylation scan of the ACTR sequence converged on two substitutions at positions 1055 and 1076 that increase affinity for both NCBD and the full length 270 kDa CBP by one order of magnitude. The first X-ray structure of the modified ACTR domain bound to NCBD revealed that the key alpha-methylated amino acids were localized within alpha-helices. Biophysical studies showed that the observed changes in binding energy are the result of long-range interactions and redistribution of enthalpy and entropy. This proof-of-concept study establishes a potential strategy for selective inhibition of protein-protein interactions involving IDPs in cells.