HSymM-guided engineering of the immunodominant p53 transactivation domain putative peptide antigen for improved binding to its anti-p53 monoclonal antibody

Abstract

A novel engineering strategy to improve autoantibody detection with peptide fragments derived from the parent antigen is presented. The model system studied was the binding of the putative p53 TAD peptide antigen (residues 46-55) to its cognate anti-p53 antibody, ab28. Each engineered peptide contained the full decapeptide epitope and differed only in the flanking regions. Since minimal structural information was available to guide the design, a simple epitope:paratope binding model was applied. The Hidden Symmetry Model, which we recently reported, was used to guide peptide design and estimate per-residue contributions to interaction free energy as a function of added C- and N-terminal flanking peptides. Twenty-four peptide constructs were designed, synthesized, and assessed for binding affinity to ab28 by surface plasmon resonance, and a subset of these peptides were evaluated in a simulated immunoassay for limit of detection. Many peptides exhibited over 200-fold enhancements in binding affinity and improved limits of detection. The epitope was reevaluated and is proposed to be the undecapeptide corresponding to residues 45-55. HSymM calculated binding free energy and experimental data were found to be in good agreement (R² > 0.75).

Keywords: Antibody; Autoantibodies; Coarse grained models; Epitope; Hidden symmetry model; Immunoassays; Paratope; Peptide engineering; Peptides; p53.

Figure 1:

Peptide Engineering Strategy. The monoclonal antibody (ab28) is specific to the decapeptide sequence corresponding to residues 46–55 of human p53 (1). Addition of flanking residues is proposed to modulate the binding affinity of the peptide by increasing (red arrows) or attenuating (blue arrows) the interaction energies of the interior residues.

Figure 2.

Peptide:Antibody Model and Construct Design. A. The PDB entry 2L14 (I) was used as the WT p53 reference structure for the target epitope, residues 46–55 (II), to generate heatmaps of the WT (III) and isolated peptide (IV). Helical wheel representations of III and IV are shown as V and VI, respectively. Paratope:epitope binding was modeled as VII and VIII. Heatmap colors indicate high (red), medium (white), and low (blue) per-residue contributions to interaction energy. B. Peptide constructs, composed of the putative epitope and C- and/or N-terminal flanking sequences, were attached to a polyethylene glycol (PEG₄) linker fused to an azido-lysine, click-ready unit. The construct shown matches 16, which has the WT flanking sequences.

Figure 3:

Comparison of Experimental and Calculated Affinities. (A) Calculated binding free energies based on the putative decapeptide epitope (residues 46–55) compared to experimentally determined values (peptides 1-9: red line, y = 0.95x + 1.80, R² = 0.71; peptides 1-24: black line, y = 1.69x + 8.30, R² = 0.24). (B) Calculated binding free energies based on the proposed undecapeptide epitope (residues 45–55) compared to experimentally determined values (peptides 1-24: y = 1.33x + 5.57, R² = 0.76). See text comments on outliers 14 and 21 (circled).