Fig. 1. Rational design of E6AP-mimicking peptides. (A) Left: X-ray crystal structure of the ternary protein complex formed by 16E6 (white), MBP (yellow) fused to the E6AP LXXLL peptide (blue), and p53 (green). PDB 4XR8. Right: interaction interface of 16E6 and the LXXLL motif of E6AP. The E6AP LXXLL peptide binds the 16E6 hydrophobic groove and drives peptide recognition. (B) Rational design of a high-affinity E6 binding peptide. The N-terminal and C-terminal lysine side chain primary amines were modified with various small molecules listed in panel (C). (C) Chemical library of small molecules used for modifying E6AP-based peptides. Planar aromatic residues are in blue, hydrophobic residues in orange, other aromatic residues in black. (D) Table of N- and C-terminally modified E6AP peptide mimics. The apparent dissociation constant KD is measured by bio-layer interferometry (BLI) in competition mode, except for *KD measured by direct BLI. (E) BLI competition assay was used to measure the binding of 6′ and 6′-3L3A. Streptavidin tips were immobilized with 1-biotin and dipped into solutions of various concentrations of analyte and 16E6. (F) Direct BLI measurement of 6′-biotin, parameters are estimated to be: kon = 1.5 × 105 M−1 s−1, koff = 4.3 × 10−3 s−1, and KD = 3.0 ± 1.8 nM.
