Structural basis for substrate recognition by the human N-terminal methyltransferase 1

Abstract

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides, respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.

Facile synthesis of SAM–peptide conjugates through alkyl linkers targeting protein N-terminal methyltransferase 1

“…24 To explore a new scaffold, we initiated our efforts to design a series of SAM–peptide conjugates by linking a SAM analog with peptides that start with either Ala or Gly through an alkyl group as our model system. Our crystal structure suggested that the distance between the sulfonium ion and the α- N -terminal nitrogen atom is 4.7 Å 30 And for most protein methyltransferases, this distance varies from 2.2 Å to 4.7 Å 29–33 Hence, we chose an ethylene or a propylene group as a linker and designed a series of compounds 1a–f targeting NTMT1 with substrate peptides that start with GPK/R. 34,35 We also designed compounds 1g–j by adding either an Ala or Gly to an ethylene group to extend the linker length for two peptide substrates PPKR and SPKR to probe an optimal linker.…”

“…29,30 So far, there is only one specific inhibitor available for this enzyme, which was synthesized through a click chemistry and reported by our laboratory. 24 To explore a new scaffold, we initiated our efforts to design a series of SAM–peptide conjugates by linking a SAM analog with peptides that start with either Ala or Gly through an alkyl group as our model system.…”

The ribosome: A hot spot for the identification of new types of protein methyltransferases

“…METTL11B was suggested to be primarily a monomethyltransferase that may prime substrates for the action of NTMT1 (55). Crystal structures of human NTMT1 in complex with substrate peptides have been solved (56,57) that support the substrate specificity of this enzyme determined from kinetic (58,59) and inhibitor studies (60). A variety of functions have been proposed for XPK N-terminal methylation of eukaryotic proteins (61), including regulating the affinity of protein binding to DNA (62,63), DNA repair (64-66) and protection from aminopeptidase attack (49).…”

Chemical Biology of Protein N‐Terminal Methyltransferases

“…This homologue was suggested as another NTMT in 2010 and confirmed to be an NTMT by 2013 . 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 .…”

“…The discovery of NTMT1/NRMT1 has led to rising reports on N‐terminal methylation existing in tumor suppressor retinoblastoma 1 (RB1), oncoprotein SET (also known as I2PP2A, TAF1α), damaged DNA‐binding protein 2 (DDB2), poly(ADP‐ribose) polymerase 3 (PARP3), and centromere proteins A and B (CENP‐A&B, Figure ) . Crystal structures of NTMT1 in complex with peptide substrates and SAH revealed the structural basis for the preferred recognition motif X‐P‐K/R (X can be any amino acid, except D or E) . Substrate recognition is discussed in more detail in Section 2.4.…”