Thermodynamic consequences of Tyr to Trp mutations in the cation-π-mediated binding of trimethyllysine by the HP1 chromodomain

Evolution has converged on cation-π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-π interaction is about −5 kcal mol −1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains. In this work, we experimentally validate that tryptophan provides the strongest cation-π binding interaction among aromatic amino acids and also lend insight into the importance of residue identity in trimethyllysine recognition by reader proteins.