Crystal Structure of SMYD2 Reveals Simultaneous Binding of PARP1 Peptides to the Active Site and an Unexpected Secondary Binding Site

Abstract

SMYD2 is a protein lysine methyltransferase with a broad substrate specificity and able to methylate both histone and non‐histone proteins such as histone H3 and H4, p53, retinoblastoma‐associated protein 1, and PTEN. However, how SMYD2 substrate specificity is precisely controlled and balanced over such a large number of targets remains elusive. Poly(ADP‐ribose) polymerase‐1 (PARP1) is another known SMYD2 substrate, acting as a first responder to DNA damage. SMYD2 mono‐methylates PARP1 at lysine 528 within the auto‐modification region. Methylated PARP1 positively regulates the poly(ADP‐ribosyl)ation activity and enhances cellular poly(ADP‐ribose) formation after oxidative stress. In this study, we determined the crystal structure of SMYD2 in complex with a K528‐containing PARP1 peptide. Unexpectedly, two peptide binding sites were identified from the structure, one corresponding to the known substrate binding site and the other a new secondary binding site. We found that the point mutations of the secondary binding site completely abolished binding of the PARP1 peptide to both sites in the isothermal titration calorimetry (ITC) analysis. The crystal structures of the corresponding SMYD2 mutants also showed disturbed binding, in which the mutation did not cause any significant structural changes but no peptide binding was observed. However, in the context of PARP1 protein, the same mutation enhanced the interaction in the ELISA assay, which was partially correlated with its substrate‐specific effects on the enzyme activities. The mutation of the secondary binding site mutation enhanced SMYD2 activity on histone H3 protein but attenuated the methylation of PARP1 protein, and there was no significant effect on histone H4 protein. Overall, our study revealed a new peptide binding site in SMYD2 that may play a regulatory role in the SMYD2 substrate selectivity.