Protein N-terminal methyltransferase 1 (NTMT1) plays an important role in regulating mitosis and DNA repair. Here, we describe the discovery of a potent NTMT1 bisubstrate inhibitor 4 (IC50 = 35 ± 2 nM) that exhibits greater than 100-fold selectivity against a panel of methyltransferases. We also report the first crystal structure of NTMT1 in complex with an inhibitor, which revealed that 4 occupies substrate and cofactor binding sites of NTMT1.
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 bisubstrate analogue
strategy is a promising approach to develop
potent and selective inhibitors for protein methyltransferases. Herein,
the interactions of a series of bisubstrate analogues with protein
N-terminal methyltransferase 1 (NTMT1) were examined to probe the
molecular properties of the active site of NTMT1. Our results indicate
that a 2-C to 4-C atom linker enables its respective bisubstrate analogue
to occupy both substrate- and cofactor-binding sites of NTMT1, but
the bisubstrate analogue with a 5-C atom linker only interacts with
the substrate-binding site and functions as a substrate. Furthermore,
the 4-C atom linker is the optimal and produces the most potent inhibitor
(K
i,app = 130 ± 40 pM) for NTMT1
to date, displaying more than 3000-fold selectivity for other methyltransferases
and even for its homologue NTMT2. This study reveals the molecular
basis for the plasticity of the active site of NTMT1. Additionally,
our study outlines general guidance on the development of bisubstrate
inhibitors for any methyltransferases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.