Abstract
Histone trimethyllysine (Kme3) reader proteins are emerging therapeutic targets. However, development of selective inhibitors has proven challenging given the conserved nature of the aromatic cage which binds Kme3 as well as the myriad reader proteins which bind Kme3 at the same position on histone tails. These readers rely on both the presence of Kme3 as well as the appropriate surrounding histone tail sequence to bind, suggesting that binding is highly cooperative. We recently found that a small subset of Kme3 readers bind with equal or tighter affinity to histone tail peptides which replace Kme3 with its neutral isostere, tBuNle. This unexpected result offers promise for therapeutic design. Herein, we utilize histone 3 tail peptides containing Kme3 or the unnatural tBuNle to probe cooperativity in reader protein binding. Through three case studies, we quantitatively determine that the degree of cooperativity in a reader protein binding histone Kme3 influences the degree of its aromatic cage preference for cationic versus neutral ligands. Moreover, we find that the degree of cooperativity differs for each reader, suggesting that such differences in cooperativity could be utilized strategically for selective inhibitor design and that mutation to either histones or readers to alter cooperativity could significantly affect a reader protein's selectivity for a specific post-translational modification.