Tuning HP1α Chromodomain Selectivity for Di‐ and Trimethyllysine

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 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.

The Role of Electrostatic Interactions in Binding of Histone H3K4me2/3 to the Sgf29 Tandem Tudor Domain

“…In addition, D877 residue of TAF3 is not directly involved in intermolecular interactions with the Kme2 side-chain, but forms a hydrogen bond with the hydroxyl group of Thr6 in the H3K4me2 peptide[ 17 ]. In contrast, E52 residue of chromodomain HP1 interacts with the positively-charged + NHMe 2 via water-mediated H-bonding[ 18 ].…”

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1

“…These data also demonstrate that N-terminal monomethylation and di/trimethylation serve non-redundant functions. There is mounting evidence of distinct functional roles for different levels of histone methylation, with the extent of methylation often controlling the binding of effector proteins [24]. There is also evidence that disruption of these levels has extreme biological effects.…”

The N-terminal methyltransferase homologs NRMT1 and NRMT2 exhibit novel regulation of activity through heterotrimer formation.

“…The shape, depth, and composition of the binding pocket, number of cation-pi interactions among the aromatic rings and polarized methyl groups, and presence of negatively charged side chains and hydrogen bond acceptors all contribute to the selectivity of distinct levels of methylation (Hughes et al, 2007;Ma & Dougherty 1997;Guo et al, 2009;Botuyan et al, 2006;Jacobs & Khorasaniz 2002;Llin et al, 2006;Taverna et al, 2007). Additionally, single amino acid substitutions within these chromodomains facilitate enough alteration of the binding pocket to differentiate between di-, and trimethylysine residues (Eisert & Waters, 2011).…”