Tuning HP1α Chromodomain Selectivity for Di- and Trimethyllysine

Graphical Abstract

Balance of powers: Mutation studies of the Drosophila HP1α chromodomain were used to determine the relative contributions of electrostatic and hydrogen-bonding interactions to the recognition of di- and trimethyllysine. The findings provide insight into how nature achieves selectivity for these two closely related post-translational modifications, and has relevance to inhibitor design as well.

Abstract

Histone lysine methylation is a critical marker for controlling gene expression. The position and extent of methylation (mono-, di-, or tri-) controls the binding of effector proteins that determine whether the associated DNA is expressed or not. Dysregulation of histone protein methylation has been associated with a number of types of cancer, and development of inhibitors for the effector proteins is becoming an active area of research. For this reason, understanding the mechanism by which effector proteins obtain selectivity for the different methylation states of lysine is of great interest. To this end, we have performed mutation studies on the Drosophila HP1α chromodomain, which binds H3K9Me₂ and H3K9Me₃ with approximately equal affinities. The selectivity of HP1α chromodomain for H3K9Me₃ over H3K9Me₂ was investigated by mutating E52 to remove or weaken the hydrogen bond to K9Me₂ while maintaining affinity for K9Me_3, including E52F, E52I, E52V, E52D, an E52Q. The E52Q mutant exhibited the greatest degree of selectivity for KMe3, with 3.5-fold weaker binding to the dimethylated peptide (K_D=52 μM) compared to the trimethylated peptide (K_D=15 μM). These studies provide insight into the role of electrostatic interactions and hydrogen bonding in the differentiation of methylation states and have implications regarding the evolutionary pressure for selectivity in this protein–protein interaction. Moreover, the information from this study may help guide inhibitor development for this class of proteins.