Substrate specificity of mammalian N-terminal α-amino methyltransferase NRMT

Abstract

N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products.

Figure 1. Substrate and product peptides affinity to NRMT measured using isothermal titration calorimetry

Wild-type 10-residue SPK-RCC1 N-terminal peptide (bottom left panel) showed efficient binding to NRMT with a K_a 1.0E5±1.10E4 M⁻¹ (K_d 10µM). A PPK-RCC1 N-terminal peptide showed very efficient binding (top left panel) with K_a1 6.60E8±2.0E8 M⁻¹ and K_a2 7.3E6±2.3E6 M⁻¹ (K_d values 1.5nM and 136nM respectively). The 10 residue me3-SPK-RCC1 (K_a 4.8E5±6.75E4 M⁻¹) and me2-PPK-RCC1 (K_a 7.70E5±1.5E5 M⁻¹) N-terminal peptides (left panel) showed efficient binding to NRMT with K_d values of 2.0µM and 1.3µM respectively.

Figure 2. NRMT can bind to its fully methylated substrate proteins

A) NRMT is co-precipitated with C-terminally FLAG-tagged substrates (RCC1 and VRK2) B) C-terminally FLAG-tagged NRMT co-precipitates with endogenous ZFP15 and Mina53. IPs were run on the 10% polyacrylamide gel and stained with Commassie blue. C) C-terminally FLAG-tagged wild type ZFP15 co-precipitates with endogenous NRMT. The PPQ non-methylatable mutant of ZFP15 does not bind endogenous NRMT. D) Both PPK- and PPQ-ZFP15 FLAG-tagged proteins are efficiently expressed and immunoprecipitated. E) N-terminal methylation activity is localized in the nucleus. NLS fused SPK-GFP3 substrates are efficiently methylated, in contrast to NES fused SPK-GFP3.

Figure 3. In vivo substrate affinity assay

A) Schematic of the in vivo substrate affinity assay B) Competitor substrate set of SPK-, SPQ- and PPK-GFP is efficiently expressed and N-terminally methylated after 32h of expression following calcium phosphate transfection. C) The efficiency of N-terminal methylation of immunoprecipitated SPK- and PPK-GFP-FLAG test substrates is variable and depends on the presence of co-transfected competitor substrate. D) Quantification of the efficiency of N-terminal methylation of SPK- and PPK-GFP-FLAG test substrates in the presence of SPK-, SPQ- or PPK-GFP competitor. Data were normalized and compared to the level of N-terminal methylation of test substrates co-expressed in the presence of unmethylatable SPQ-GFP competitor substrate by two-tailed independent t tests. * indicates P<0.01. n=3 independent repetitions per combination. Error bars: +/− 1 s.d.

Figure 4

A) NRMT is inhibited in vitro by me3SPK and by me2PPK product peptides with variable efficiency dependent on the affinity of the enzyme for the substrate. B) Quantification of inhibition by me3SPK and me2PPK product peptides. Data were normalized and compared to the level of N-terminal methylation of SPK-RCC1 or PPKRCC1 substrates methylated by recombinant NRMT in the presence of 0.05µM me3SPK or me2PPK product peptide by two-tailed independent t tests. P-value is <0.01, n=2 independent repetitions per reaction. Error bars: +/− range.