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 
H3K9Me(2) and H3K9Me(3) with approximately equal affinities. The selectivity of 
HP1α chromodomain for H3K9Me(3) over H3K9Me(2) was investigated by mutating E52 
to remove or weaken the hydrogen bond to K9Me(2) 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.