Mechanistic insights into allosteric regulation of methylated DNA and histone H3 recognition by SRA and SET domains of SUVH5 and the basis for di-methylation of lysine residue.

Su-(var)3-9 homologue 5 (SUVH5), a member of SUVH family of histone lysine methyltransferase (HKMT) in Arabidopsis, is involved in epigenetic regulation of chromatin by recognizing 5-methyl-cytosine (5mC), in both CpG and non-CpG DNA context, through SRA domain and simultaneously performing the di-methylation of lysine 9 of histone H3 (H3K9) through SET domain. Here, we establish that the SET domain of SUVH5 allosterically restricts the SRA domain to the 5mC containing strand(s) of fully methylated CpG, hemi-methylated CpG and methylated CpHpH DNA. In addition, SET domain enhances the binding affinity of the SRA-SET dual domains to fully-mCpG but not to hemi-mCpG. Also, the recognition of methylated DNA by the SRA positively influences the recognition of H3K9 by the SET domain. Our further studies revealed that the SET domain recognizes the "A(R/K)KST" motif present in H3K9 and in other histone H2A variants. Further, computational analyses and quantum mechanics/molecular mechanics calculations explain the bases for robust mono-MTase but weak di-MTase activities of SUVH5. Given that the majority of eukaryotic proteins, including those involved in epigenetic gene regulation, contain more than one domain, our study suggests that understanding the allosteric regulation among multiple domains of proteins is relevant for unravelling biological outcomes.