Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of its binding to substrate peptides

Abstract

PRMT1 is the predominant type I protein arginine methyltransferase in mammals and highly conserved among all eukaryotes. It is essential for early postimplantation development in mouse. Here we describe the crystal structure of rat PRMT1 in complex with the reaction product AdoHcy and a 19 residue substrate peptide containing three arginines. The results reveal a two-domain structure-an AdoMet binding domain and a barrel-like domain-with the active site pocket located between the two domains. Mutagenesis studies confirmed that two active site glutamates are essential for enzymatic activity, and that dimerization of PRMT1 is essential for AdoMet binding. Three peptide binding channels are identified: two are between the two domains, and the third is on the surface perpendicular to the strands forming the beta barrel.

Figure 1. Members of the PRMT1 Family

(A) Structure-based sequence alignment of rat PRMT1, PRMT3 [43], and yeast RMT1 [44]. Letters (A–Z) for helices and numbers (1–15) for strands indicate the secondary structure elements of rat PRMT1 or PRMT3; residue numbering is shown above or below the sequences. The color coding is red for the N terminus including helix αY (residues 41–51), green for the AdoMet binding domain (residues 52–176), yellow for the β barrel structure (residues 177–187 and 217–352), and blue for the dimerization arm (residues 188–216). For rat PRMT1, the amino acids highlighted (white against black) are invariant among PRMT1s from various organisms: rat PRMT1 (GenBank accession number NP_077339), human PRMT1-v2 (modified from CAA71764), PRMT1-like (also called HMT1L3, XP_006990), C. elegans PRMT1 (CAB54335), D. melanogaster PRMT1 (AAF54556), A. thaliana PRMT1 (CAB79709), A. thaliana PRMT1-like (AAC62148), S. pombe PRMT1 (CAB63498), and S. cerevisiae RMT1 (585608). The Xenopus and zebrafish PRMT1 sequences were assembled from ESTs with sequences highly similar to that of rat PRMT1. The N-terminal sequences shown with a strikethrough are not observed in the crystal structures, presumably due to being highly disordered in the crystals. The asterisks above the sequence indicate residues important for cofactor binding (see Figure 4A) and/or catalysis (see Figure 7A). (B) N-terminal splicing variants of human PRMT1.

Figure 2. Activity of PRMT1 Proteins

(A) Reactions (20 μl) contained 4 or 8 μM of purified hnRNP A1 (130 or 260 μg/ml), 40 μM [methyl-³H]AdoMet (0.5 μCi), and the indicated amount of PRMT1 in 100 mM Tris (pH 8.0), 200 mM NaCl, 2 mM EDTA, and 1 mM dithiothreitol. After incubating at 37°C for 15 min, the samples were analyzed by SDS-PAGE. The upper panel shows the Coomassie-stained gel of total PRMT1 and hnRNP A1, while the lower panel shows the image resulting from fluorography. Various amounts of wild-type PRMT1 (lanes 3–5, 11–15, and 21–23) were used so that the relative activity of mutant PRMTs could be estimated. (B) Methylation of peptides R3 or R1 (RGG) and competition by hnRNP A1. Reaction conditions are identical to Figure 2A and include 10 μg/ml of PRMT1, 5 μM of hnRNP A1 and/or various amounts of peptides as indicated. (C) Time course of AdoMet crosslinking of wild-type PRMT1 and ΔARM mutant. (D) AdoMet crosslinking of various PRMTs and mutants. The samples were treated with UV for 1 hr. Controls were used: yRMT1 (yeast RMT1), PRMT3 (full-length rat PRMT3), PRMT3Δ200 (rat PRMT3 conserved core), and HhaI (DNA cytosine methyltransferase).

Figure 3. Structure of PRMT1

(A) Two views (top and bottom panels) of monomer structure. The N-terminal helix αY is shown in red, and the AdoMet binding domain in green. The bound AdoHcy is shown in a stick model with the sulfur atom (where the transferable methyl group would be attached in AdoMet) shown in yellow. The β barrel structure is shown in yellow, and the dimerization arm (which is inserted into the β barrel) is in light blue (see Figure 1A). The bound arginine (blue) in the S14-AdoHcy-R3 ternary complex defines the active site, located between the AdoMet binding domain (green) and the β barrel (yellow). (B) Superposition of PRMT1 (residues 41–353, colored according to Figure 1A) and PRMT3 core (residues 208–528, in gray). Besides deletion or insertion (located in loops between β10 and β11 and between β14 and β15), the two structures can be superimposed with less than 1 Å of root-mean-square deviation between them.

Figure 4. Stereo View of AdoHcy Binding

A) In PRMT1, the difference electron density map is contoured at 5.5σ. Dashed lines indicate hydrogen bonds. (B) In PRMT3 (PDB ID code 1F3J; [43]), the corresponding N-terminal residues F218, Y217, and Y221 (red) are not observed in the PRMT1 structure.

Figure 5. Dimer Formation of PRMT1

(A) Two ring-like dimers (related by a crystallographic 2-fold) connected by a surface C254 via a disulfide bond are shown with a difference electron density map contoured at 5.5σ. The dimer is formed through the arm (blue) and the outer surface of AdoMet binding domain (green), as indicated. The AdoHcy (gray) is in a stick model. (B) Two opposite GRASP surfaces of PRMT1 dimer. The surface is colored red for negative, blue for positive, and white for neutral. (C) Two opposite GRASP surfaces of PRMT1 monomer. Dimerization is mediated through hydrophobic patches of the arm and the outer surface of AdoMet binding domain, as indicated.

Figure 6. Structure of Ternary Complex of S14-AdoHcy-R3 Peptide (Sequence Shown at the Top)

(A) Solvent-accessible molecular surface with bound AdoHcy and Arg shown as stick models and indicated by the arrows. In the absence of helix αX (see Figure 3B), which is disordered in the PRMT1 crystals, AdoHcy is exposed and readily visible. The three discontinuous peptide binding sites P1, P2, and P3 are shown as green tubes. (B) Ribbon representation of (A) with the three peptide binding sites shown as annealed omit electron densities (black) contoured at 5.0σ. Some of the structural elements flanking sites P2 and P3 are labeled. (C) A 90° rotation of (B) showing that the binding sites P1 and P2 can be connected so that the middle Arg9 of peptide R3 is bound at the active site. (D) Two side views of (A) showing the three peptide binding sites as well as other acidic grooves parallel to P3 (indicated by the arrows). The acidic residues flanking these grooves are labeled. In the left panel, it can be seen that connecting P2 and P3 would place the terminal arginine (Arg3 or Arg15) at one end of P2 in the active site.

Figure 7. Active Sites of PRMT1 and PRMT3

(A) PRMT1 active site with bound Arg in stereo. The annealed omit electron density map, contoured at 5.0σ of the arginine, is shown as an insert. (B) Superimposition of PRMT1 and PRMT3 (PDB ID code 1F3J) active sites in stereo. Only the PRMT3 residues are labeled. The arrow indicates transfer of the methyl group (attached to AdoHcy) to the bound Arg. (C) pH dependence of PRMT1 and PRMT3 activities. Reactions (20 μl) contained 5 μM of purified hnRNP A1 or 100 μM of R3 peptide, 10 μg/ml of PRMT1 or 50 μg/ml of PRMT3Δ200, 40 μM [methyl-³H]AdoMet (0.5 μCi) in 100 mM buffer, 200 mM NaCl, 2 mM EDTA, and 1 mM dithiothreitol. The buffers used were sodium acetate (pH 4 and 5), MES (pH 6.0, 6.3, 6.5, and 6.8), HEPES (pH 7.0 and 7.5), Tris (pH 8.0 and 8.5), and glycine (pH 9.0, 9.5, and 10.0). After incubating at 37°C for 15 min, 2.5 μl of 100 mg/ml BSA was added, followed by 0.5 ml of 20% TCA. The samples were filtered and washed three times with 20% TCA through a GF/F filter (Millipore), dried, and subjected to liquid scintillation counting.