Fluorine-Thiol Displacement Stapling on the Disordered α_B of pKID Domain Increases Its Helicity and Affinity to KIX

The development of high-affinity ligands specifically targeting intrinsically disordered protein interactions has remained challenging due to the lack of well-defined binding pockets and shallow binding surfaces commonly found at their interfaces. Here, we employed our fluorine-thiol displacement reaction (FTDR) peptide-stapling platform to synthesize a library of peptide-based ligands derived from the α_B-helix of the disordered pKID to target its binding partner KIX. Our library revealed that helical formation and affinity to KIX is highly favored when the α_B peptide was stapled at sites corresponding to Arg¹³⁵ and Ser¹⁴², further supporting the hypothesis that stabilization of α_B significantly influences the overall binding affinity of pKID to KIX. We also found that the highest binding peptide, α_B-RSpS, may form secondary contacts at the MLL site on KIX in addition to binding at the primary pKID site. Lastly, no binding to KIX was observed for any α_B-stapled peptide that lacked the conserved helix-flanking prolines Pro¹³² and Pro¹⁴⁶. Conserved helix-flanking prolines have previously been shown to modulate the binding affinities of other disordered domains in other proteins including MLL and p53. However, to our knowledge this is the first evidence within α_B of pKID.