Directed Evolution of the Cbx1 Chromodomain Generates a High-Affinity Trimethyllysine-Binding Variant

Directed evolution is a powerful and versatile strategy for engineering proteins with enhanced affinity and tailored molecular recognition properties. Here, we applied phage display-guided directed evolution to enhance the binding of the wild-type Cbx1 chromodomain (Cbx1-CD) to an H3K9me3-containing peptide. Screening a soft-randomized library targeting residues surrounding the aromatic cage yielded C29, which bound the H3K9me3 peptide with a KD of 57 nM and exhibited an approximately 14-fold lower dissociation rate constant than Cbx1-CD. Across an H3K9me3-derived alanine-scanning series and a peptide panel, C29 showed stronger binding signals than Cbx1-CD for multiple peptides containing trimethyllysine (Kme3), suggesting lower flanking-sequence dependence for C29 than for Cbx1-CD. An AlphaFold3 model of the C29-H3K9me3 complex suggested local changes in the hydrophobic and electrostatic environment surrounding the Kme3-binding pocket, providing a mechanistic hypothesis for C29's enhanced affinity and reduced flanking-sequence dependence. When two Kme3-containing peptides were spiked into a 100-fold molar excess of unmodified background peptides, C29-conjugated beads effectively enriched both Kme3 peptides relative to the unmodified background. These findings define C29 as a high-affinity Kme3-binding Cbx1 chromodomain variant and support its further evaluation as an affinity reagent for Kme3-containing peptide enrichment.