Enzyme. Reasoning that the MTase would likely bind extra avidly to its substrate than its product, we performed an MS-based quantitative interaction peptide pull-down screen19 employing a synthetic biotinylated peptide corresponding to Nterminally unmodified eEF1A sequence as bait, plus the corresponding N-terminally trimethylated peptide as reference, to enrich interacting proteins from a human cell extract (Fig. 1a). Proteins binding to the immobilized peptides have been digested with trypsin, and the resulting peptides had been analyzed by state-of-theart nanoflow liquid chromatography tandem mass spectrometry (LC-MSMS), followed by protein quantification employing the MaxLFQ algorithm20 embedded in MaxQuant software suite21. In total, 157 proteins were identified to be significantly enriched by the unmethylated bait and 174 proteins by its methylated counterpart (Fig. 1b, Supplementary Fig. 1, and Supplementary Data 1). Importantly, peptide pull-downs intrinsically enrich proteins that biophysically interact with the bait peptide in vitro and, consequently, not all hits in such screens are necessarily biologically relevant. Interestingly, the Telenzepine Autophagy putative methyltransferase METTL13 was amongst the proteins most strongly enriched by the unmodified bait peptide and was consequently selected for additional characterization (Fig. 1b). METTL13 harbors two distinct predicted MTase domains that each belong to the 7BS superfamily (Fig. 1c and Supplementary Fig. 2). The N-terminal domain (right here denoted MT13-N) belongs to a not too long ago discovered enzyme loved ones consisting of most likely KMTs15 plus the C-terminal domain (right here denoted MT13-C) lacks close paralogs, but is distantly related to spermidine synthase (SpdS) (Fig. 1c). We expressed and purified human MT13-N and MT13-C individually as recombinant proteins from E. coli and assessed their capability to methylate recombinant eEF1A in vitro. As the conformation of eEF1A is dependent on nucleotide binding22 and we’ve previously observed that the efficiency of other eEF1A-specific MTs could be modulated by the addition of guanosine nucleotides16,23, the experiments were performed inside the presence of GDP, GTP, or without the need of exogenously added cofactors. Furthermore, we evaluated eEF1A1 with an affinity tag positioned at either the N or C terminus as substrate. Importantly, N-terminal methylation of human eEF1A happens on Gly2 following enzymatic removal of the iMet, along with the endogenous methionine aminopeptidase in E. coli is predicted to process heterologously expressed human eEF1A accordingly24. These experiments Thiodicarb Purity & Documentation revealed that each MTase domains of METTL13 had been capable of methylating eEF1A in vitro and that their activities were clearly distinct. MT13-N methylates eEF1A1 irrespective of affinity tag placement at the N or C terminus, and methylation was inhibited by the addition of nucleotides (Fig. 1d). Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A had been made use of as baits to enrich proteins from HAP-1 cell extracts. b Volcano plot demonstrating enrichment of proteins by the unmodified (cyan circles) versus N-terminally trimethylated (magenta circles) bait peptides. The curved line represents the significance cutoff (FDR = 0.01 and s0 = 0.1). The putative methyltransferase METTL13 is indicated and all represented proteins are listed in Supplementary Data 1. c Domain organization of METTL13. The boundaries for made use of constructs encompas.
