Abstract
α-N-terminal methylation of proteins is an important post-translational modification that is catalyzed by two different N-terminal methyltransferases, namely NTMT1 and NTMT2. Previous studies have suggested that NTMT1 is a tri-methyltransferase, whereas NTMT2 is a mono-methyltransferase. Here, we report the first crystal structures, to our knowledge, of NTMT2 in binary complex with S-adenosyl-L-methionine as well as in ternary complex with S-adenosyl-L-homocysteine and a substrate peptide. Our structural observations combined with biochemical studies reveal that NTMT2 is also able to di-/tri-methylate the GPKRIA peptide and di-methylate the PPKRIA peptide, otherwise it is predominantly a mono-methyltransferase. The residue N89 of NTMT2 serves as a gatekeeper residue that regulates the binding of unmethylated versus monomethylated substrate peptide. Structural comparison of NTMT1 and NTMT2 prompts us to design a N89G mutant of NTMT2 that can profoundly alter its catalytic activities and product specificities.
Highlights
Cheng Dong 1, Guangping Dong[2], Li Li1, Licheng Zhu[1,3], Wolfram Tempel[1], Yanli Liu[1], Rong Huang2 & Jinrong Min[1,4] α-N-terminal methylation of proteins is an important post-translational modification that is catalyzed by two different N-terminal methyltransferases, namely NTMT1 and NTMT2
NTMT1 has been classified as a tri-methyltransferase[12,19,20], whereas NTMT2 was reported as a mono-methyltransferase[18]
We determined the crystal structure of NTMT2 in ternary complex with cofactor SAH and a substrate peptide SPKIRA, which is consistent with its classification as a SAM-dependent class I methyltransferase[22]
Summary
Cheng Dong 1, Guangping Dong[2], Li Li1, Licheng Zhu[1,3], Wolfram Tempel[1], Yanli Liu[1], Rong Huang2 & Jinrong Min[1,4] α-N-terminal methylation of proteins is an important post-translational modification that is catalyzed by two different N-terminal methyltransferases, namely NTMT1 and NTMT2. Loss of N-terminal methylation of regulator of chromatin condensation 1 (RCC1) diminishes its binding affinity for DNA, and results in defects of spindle assembly and chromosome segregation[8]. In 2013, another human NTMT, NTMT2/METTL11B/NRMT2, was described as a monomethyltransferase[18], NTMT1 is able to catalyze trimethylation[12,19,20] We and another group previously solved crystal structures of NTMT1 in ternary complex with SAH and substrate peptides, and proposed a catalytic mechanism[13,21]. In order to unravel the molecular basis of the substrate and product specificity of NTMT2, we determine the X-ray crystal structures of NTMT2 in binary complex with SAM, as well as in ternary complex with SAH and an RCC1-derived peptide (SPKRIA). The N89G mutant of NTMT2 is more active than the wild type NTMT2, which is able to convert S/APKRIA peptides from the monomethylation state to di-/tri-methylation states in our in vitro enzymatic assays
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