ABSTRACT
Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Lysine/analogs & derivatives , Mutagenesis , Animals , Chromosomal Proteins, Non-Histone/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Histones/chemistry , Histones/metabolism , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , Protein Binding , Thermodynamics , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
Fluorophenylalanines bearing 2-5 fluorine atoms at the phenyl ring have been genetically encoded by amber codon. Replacement of F59, a phenylalanine residue that is directly involved in interactions with trimethylated K9 of histone H3, in the Mpp8 chromodomain recombinantly with fluorophenylalanines significantly impairs the binding to a K9-trimethylated H3 peptide.
