Structural Basis For Peptide Binding Of α-N Terminal Methyltransferase From Saccharomyces Cerevisiae

Post-translational modifications refer to chemical modifications that are essential for mature biological function of proteins.Proteins from eukaryotic origins undergo various post-translational modifications i.e.ubiquitination,SUMOylation and methylation.The first reported N-terminal methylation modification of proteins occurred on bacterial ribosomal proteins L16 and L33.Subsequently,many proteins such as regulator of chromatin condensation 1(RCC1),centromere protein A/B(CENPA/B)and damaged DNA-binding protein 2(DDB2)have been reported to undergo Nterminal methylation.Overall,N-terminal methylation involves a wide range of functions in cellular process,i.e.mitotic,development progression and DNA damage repair.ScNTM1 is an α-N terminal methyltransferase encoded by YBR261C/Tae1 gene in Saccharomyces cerevisiae(herein referred to as ScNTM1),which methylates ribosomal proteins Rp112ab and Rps25a/Rps25b.It is also the first reported enzyme that can methylate the α-N terminal of ribosomal protein in eukaryotes.Previous knockout studies demonstrated that the methylated ribosomal proteins might protect yeast from microbial attack and play an important role in protein synthesis.However,few studies have been reported on the biochemistry of ScNTM1 from yeast and its molecular mechanism of interaction with substrates.To elucidate the molecular interactions between the ScNTM1 and its substrate,the crystal structures of ScNTM1 from Saccharomyces cerevisiae in complex with SAH and Rps25a-derived hexapeptide(PPKQQL)as well as the binary complex of ScNTM1-SAH were obtained.Structure analysis revealed that ScNTM1 adopted a core methyltransferase fold the same as other solved SAM-MTases for SAH-binding.Some structural changes on the surface of ScNTM1 display evolution-related variations.Compared to SAH-NRMT1,most of the residues in the core domain of ScNTMl that provide hydrophobic interactions for SAH binding are conserved,such as Gly73,Phe98,Gly122 and Leu 145.As Pro94 replaces the corresponding Ile in NRMT1,the adenine ring deflects about 20°to adapt to the ring-ring stacking between adenine and Pro94.By comparing the ternary complex structure of ScNTM1 and human NRMT1 and NRMT2,we found that most of the residues involved in the binding of Rps25a substrate are conserved.The key residues participating in the binding of the first two amino acids of the substrate to the protein are the same,such as Asn172 and Trp140.The last four amino acids that interact with the protein have changed due to the different swing directions of the substrate polypeptide.The hydrogen bond between Q4 and the side chain of Tyr32 is unique to ScNTM1,and this hydrogen bond plays an important role in the recognition of P2 and Q4 in polypeptides.The key residues involved in the binding of Rps25a and SAH were verified by point mutation and isothermal titration calorimetry(ITC)experiments in vitro.Structural alignment of the ternary and binary complex indicate a slight shift occurring in some regions i.e.residues 25-36,residues 183-186 and residues 215-221 upon peptide binding.These studies have enriched our understanding of the selectivity and specificity of α-N-terminal methyltransferase substrates.