Molecular Basis And Functional Studies Of Histone ?-N-Terminal Methylation By NRMT1

Histone methylation is an important modification in epigenetic regulations. Well studied histone lysine and arginine methylations are considered to take indispensable parts in gene regulation and transcription, abnormities of which are related to the occurrence of various kinds of cancers as well as mental retardations. In contrast, it was not until the first eukaryotic protein ?-N-methyltransferase, NRMT1 was discovered did the investigations on histone ?-N-methylation begin to make its own breakthrough. Unlike most histone methyltransferases, NRMT1 is able to methylate both histone and non-histone substrates, indicating its important and diverse roles in biological functions. Despite of the above mentioned, little is known about how NRMT1 recognize and catalyze its substrates.To decipher these unsolved questions, we solved crystal structures of NRMT1/CENP-A/SAH and NRMT1/DmH2B/SAH complexes, and elucidated molecular mechanism of NRMT1 recognition motif “Xaa-Pro-Lys/Arg”(where Xaa denotes small residues). Detailed structural analysis revealed NRMT1 consists of a typical SAM-MTase core domain, which resembles lysine methyltransferase DOT1 L and arginine methyltransferase PRMT7, but not SET-domain containing histone methyltransferases. SAM binding sites are well conserved among these enzymes, NRMT1 lid module is essential in substrate binding. A methyltransfer kinetic model was proposed based on the structure and biochemical assays. Bioinformatics analysis reveals NRMT1 homologs exist from lower eukaryotes to higher eukaryotes; superimposition of the two structures confirmed high conservation properties among NRMT1 homologs.In vitro biochemical studies confirmed important residues in substrate recognition and catalysis, revealing a processive manner of NRMT1 in methylation. Further cellular studies discovered the essential role of ?-N-terminal tri-methylation of CENP-A on mitosis,abnormalities of which would results in the formation of the micronucleus, and further lead to cell division defects. We also solved the crystal structure of NRMT2(an NRMT1 homolog) in complex with SAH and N-terminal mono-methylated CENP-A. Structural analysis revealed great resemblance between NRMT1 and NRMT2; SAH and substrate binding sites are well conserved. In vitro biochemical studies suggested NRMT2 catalyze in a distributive manner, with the catalytic efficiency much lower than that of NRMT1.Application of Analytical Ultracentrifugation confirmed both NRMT1 and NRMT2 catalyze methyltransfer reaction as monomers in solution.In all, for the first time our study reported the molecular basis for histone ?-N-methylation by NRMT family members, which shed lights on other protein ?-N-methyltransferases. In addition, the crystal structure of NRMT1 complex reaches a high 1.2(?), which provides a solid foundation for lead optimization and tool compound development in cell division related studies.