Work

Site-Specific Immobilization of ZNRF3 Reveals the Importance of Target Structural Integrity on Macrocyclic Peptide Selections

Year: 2026

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide Phage display of macrocyclic peptide libraries has proven highly effective for ligand discovery, yet the impact of target immobilization and structural integrity on selection outcomes has not been systematically examined. Using the ZNRF3 ectodomain as a model, we incorporated p-azidophenylalanine (AzF) at three phenylalanine residues with distinct solvent exposures (F217, F85, F156) to enable selective perturbation of the protein’s structure via site-specific strain-promoted azide–alkyne cycloaddition (SPAAC) immobilization. Biochemical evaluation of the mutants confirmed efficient conjugation and structural disruption of the protein, with the F156AzF mutant displaying the most significant reduction in activity. Phage selections using a CX 12 C macrocyclic library demonstrated that enrichment efficiency and sequence diversity correlated with structural preservation: F217AzF and F85AzF yielded robust and overlapping peptide pools, while F156AzF produced few and modestly enriched sequences. Biophysical characterization of top hits indicated that peptides derived from structurally intact immobilizations were most likely to bind wild-type ZNRF3, with the highest-affinity ligand, 85–2 ( K D = 124 nM), emerging from the shared pool. This work reports a technique to selectively disrupt protein domains during phage selections, while also demonstrating that the structural integrity of immobilized targets is a primary determinant of phage display success. Not only does the necessity to maintain structural integrity influence sequence composition and affinities of peptides toward native protein targets, but also the overall enrichment efficiency of the selection itself. While disruptive immobilization may still yield useful ligands, strategies that preserve native folds enhance phage enrichment and maximize the identification of biologically relevant binders.

Source: ACS Chemical Biology

DOI: 10.1021/acschembio.6c00015