Molecular Basis For CENP-B Methylation By ?-N-methyltransferase NRMT1

CENP-B locates in the central domain of CENPs complex and binds to centromeric heterochromatin.The abnormal expression of CENPs may lead to the disassembly of centromeres due to the breakdown of ordered DNA-protein complex structure,resulting in abnormal chromosomal separation and genomic instability.At present,CENP-B that bind to chromatin are mainly in the form of trimethylated ?-N terminal.Furthermore,it has been shown that the N-terminal methylation of CENP-B can enhance the binding ability of the DNA binding domain with the 17bp DNA on the centromere and maintain the activity of the centromere.However,the detailed structural mechanism of how the amino terminal methylation of CENP-B can influence the bind ability to DNA is still in the blank.N-terminal RCC1 Methyltransferase 1(NRMT1)is the only methyltransferase that can catalyze trimethylation of ?-N-terminal in human.The functions of NRMT1 are diverse and important.And its substrates include both histones and non-histones,and also include centromere protein CENP-B,which we are concerned about.There have been many reports on the identification of CENP-B as its substrate,but how NRMT1 recognizes and catalyzes CENP-B is not well understood.To solve the problems above,we first expressed and purified NRMT1 protein.Crystallography was used to grow and optimize the complex crystals of NRMT1,CENP-B polypeptide substrate and SAH.And the crystal structure of high resolution is solved.By using in vitro binding assay and enzyme activity assay,we confirmed the interaction between NRMT1 and CENP-B substrate and the catalytic capacity of NRMT1 to CENP-B substrate.In addition,biochemical studies of the mutant protein and structural studies revealed the important residues of NRMT1 to bind and catalyze CENP-B.Finally,the molecular mechanism of NRMT1 recognition and catalytic N-terminal trimethylation of CENP-B was clarified by analyzing a large number of biochemical experimental results and structural details.Our study not only provides supporting evidence for previous functional studies,but also provides effective molecular basis for future research on target drug precursors or small molecule tools to CENP-B.