Abstract
Allosteric regulation allows proteins to dynamically respond to environmental cues by modulating activity at sites away from the catalytic center. Despite its importance, the SET-domain protein lysine methyltransferase superfamily has been understudied. Here, we present four crystal structures of SMYD2, a unique family member with a MYND domain. Our findings reveal a novel allosteric binding site with high conformational plasticity and promiscuity, capable of binding peptides, proteins, PEG, and small molecules. This site exhibits positive cooperativity with substrate binding, influencing catalytic activity. Mutations here significantly alter substrate affinity, changing the enzyme's kinetic profile. Specificity studies show interaction with PARP1 but not histones, suggesting targeted regulation. Interestingly, this site's function remains unaffected by active site changes, indicating unidirectional mechanisms. Our discovery provides novel insights into SMYD2's biochemical regulation and lays the foundation for broader research on allosteric control in lysine methyltransferases. Given SMYD2's role in various cancers, this work opens exciting avenues for designing specific allosteric inhibitors with reduced off-target effects.