An asparagine/glycine switch governs product specificity of human N-terminal methyltransferase NTMT2

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.

The N‐terminal methyltransferase homologs NRMT1 and NRMT2 exhibit novel regulation of activity through heterotrimer formation

“…We next wanted to determine which domains on NRMT1 and NRMT2 are responsible for heterotrimer formation. The crystal structures of NRMT1 and NRMT2 (PDB codes 2EX4 and 5UBB) have been determined to 1.75 and 2 Å, respectively, and as predicted by homology modeling, are almost identical barring the additional N‐terminal domain on NRMT2, which was not included in the structure . The core of both enzymes is a seven‐stranded β sheet (β1‐7) flanked by five α‐helices (α1‐5) .…”

Chemical Biology of Protein N‐Terminal Methyltransferases

“…NTMT2 shares a comparable substrate recognition mode to that of NTMT1. A structural comparison of the substrate‐binding region of NTMT2 with that of NTMT1 revealed a similar set of residues (Figure ) . In NTMT2, key substrate‐engaging residues are Asn223, Trp191, Asp232, and Asp235, which are in agreement with Asn168, Trp136, Asp177, and Asp180 in NTMT1 .…”

“…A structural comparison of the substrate‐binding region of NTMT2 with that of NTMT1 revealed a similar set of residues (Figure ) . In NTMT2, key substrate‐engaging residues are Asn223, Trp191, Asp232, and Asp235, which are in agreement with Asn168, Trp136, Asp177, and Asp180 in NTMT1 . NTMT2 is able to methylate XPKRIA hexapeptides, although its physiological substrate has yet to be identified.…”

“…Both NTMT1 and NTMT2 recognize an X‐P‐K/R consensus sequence, in which X can be any amino acid, except D or E . Although NTMT2 was originally proposed to be a monomethylase, recent studies indicated that it could also fully methylate both GPKRIA and PPKRIA peptides . However, the physiological substrate of NTMT2 remains to be uncovered.…”

“…YBR261C and NTMT1/2 are able to methylate nona‐ and hexamer peptides, respectively. In addition, eukaryotic protein NTMTs share an X‐P‐K/R motif.…”