INVESTIGATION INTO THE BINDING MECHANISMS OF HISTONE READER PROTEIN RECOGNITION OF POST-TRANSLATIONAL MODIFICATIONS

Histone post-translational modifications (PTMs) are well-established to play roles in regulation of gene expression and other key cellular processes. The mechanisms through which these PTMs exert their function is in part through recognition by histone reader proteins. These reader proteins bind to histone PTMs in a sequence-selective manner, dictating downstream effects. Reader protein dysregulation has been shown to lead to diseases, including many types of cancer. Thus, many histone reader proteins have emerged as therapeutic targets. Despite their biological roles, the development of effective inhibitors for many classes of histone reader proteins has proven challenging due to their shallow, surface-exposed binding pockets as well as difficulty in selectively targeting one reader protein over others in the same family. This thesis uses biochemical and biophysical techniques to interrogate reader protein binding of multiple histone PTMs, offering an enhanced understanding of these complex molecular recognition events and the noncovalent interactions which drive them. Improved understanding of these binding events has the potential to inform and enable selective inhibitor design for these reader proteins.Specifically, we broadly evaluated the charge dependence in binding of trimethyllysine (Kme3) reader proteins, finding unexpectedly that many proteins are capable of binding with equal or tighter binding to a neutral isostere of Kme3. We find that binding to Kme3 is driven by cation-π interactions while binding to the neutral isostere is driven by the hydrophobic effect. Differences in charge dependence in binding between highly similar proteins were further explored to evaluate several factors that contribute to that phenomenon. Additionally, we evaluated binding of asymmetric dimethylarginine by the SPIN1 Triple Tudor Domain, reporting for the first time that an electrostatically tunable CH3-π interaction contributes significantly to binding. We also explored binding of the AF9 YEATS domain to various histone acyllysine PTMs as well as to non-natural isosteres, finding that amide-π and CH-π interactions contribute substantially to binding. Lastly, we evaluated binding of WDR5 to histone serotonylation and isosteres, finding that an edge-face π-π interaction is critical to binding, but is not dependent on the electrostatic character of the face. In sum, this thesis provides insight into binding mechanisms by many therapeutically relevant proteins and also provides quantitative assessment of many different types of noncovalent interactions critical to protein-protein interactions.