Authors
Shuhao Li, Dingyi Wang, Binyi Yang, Guosong Dong, Huadong Liu, Bing Wang, Lei Li
Published in
ACS chemical biology. Oct 29, 2026. Epub Oct 29, 2026.
Abstract
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.
PMID:
42950181
Bibliographic data and abstract were imported from PubMed on 29 Oct 2026.
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