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

Abstract

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) based chemical stapling platform to synthesize a library of peptide-based ligands derived from the αB 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 αB peptide was stapled at sites corresponding to Arg135 and Ser142, further supporting the hypothesis that stabilization of αB significantly influences the overall binding affinity of pKID to KIX. We also found that the highest binding peptide, αB-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 αB 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 αB of pKID.