Negative Cooperativity in the UHRF1 TTD‐PHD Dual Domain Masks the Contributions of Cation‐π Interactions between Trimethyllysine and the TTD Aromatic Cage

UHRF1 is a promising epigenetic target in oncology, but inhibitor development has proven challenging due to the interplay between its tandem Tudor domain (TTD) and plant homeodomain (PHD). The TTD binds trimethyllysine (Kme3) at position 9 while the PHD binds Arg at position 2 on histone 3. Herein, we report how the PHD influences TTD recognition of the histone 3 tail containing Kme3 (H3K9me3) versus its neutral isostere, tert-butyl norleucine (tBuNle). Our findings show that the dual domain binds both peptides equally, supporting tBuNle's potential for inhibitor development. However, unexpectedly, the binding mechanism of H3K9me3 differs between the single and dual domains. In the TTD alone, Kme3 is bound in the aromatic cage via electrostatically tunable cation-π interactions, but in the dual domain, Kme3 binding is independent of electrostatics in the aromatic cage-an unprecedented observation. Computational studies suggest cation-π interactions should contribute in both cases. The contrasting experimental and computational results point to an unusual example of negative chelate cooperativity: interactions between the histone and PHD mask the mechanism of TTD recognition of K9me3. This work underscores the complexity of histone post-translational modification (PTM) readout in multi-domain proteins and demonstrates the first example of a masked cation-π interaction.