An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation

Abstract

Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N₃ methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases-human SETD6 and the plant LSMT-but differs in the residues constituting the active site. In the SETD3 active site, Asn²⁵⁵ engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (k_cat/K_m ) on histidine than on lysine. Here, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn²⁵⁵ with phenylalanine (N255F), together with substitution of Trp²⁷³ with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms-the N₃ atom of histidine and the terminal ϵ-amino nitrogen of lysine-occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn²⁵⁵ substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.

Keywords: S-adenosylmethionine (SAM); enzyme catalysis; enzyme kinetics; glutamine methylation; histidine; histidine methylation; lysine methylation; protein methylation; structural biology.

Figure 1.

Comparison of active sites of SETD3, SETD6, and LSMT. A, SETD3 Asn²⁵⁵ makes a hydrogen bond with the protonated N₁–H of His⁷³ of actin. B–D, the mutant N255F is not active on His⁷³ peptide. The assays were performed at 37 °C and pH 8.0 using WT = 0.18 μm (20 min), N255A = 0.18 μm (20 min), N255V = 0.72 μm (20 min), and N255F = 3 μm (3 h). Data represent the mean ± S.D. of two independent determinations (n = 2) performed in duplicate. E, a model of Phe at residue 255 clashes with Trp²⁷³. F, structure-based sequence alignment of SETD3, SETD6, and LSMT. The four active-site residues in SETD3 are highlighted in red; invariant residues in blue, conserved residues in gray. G, Tyr³¹² is the only conserved active-site residue among the three enzymes.

Figure 2.

Kinetics of SETD3 and the N255F/W273A variant on His⁷³ and Lys⁷³ peptides. A–C, comparison of WT and the mutant on peptides of (A) His⁷³ (residues 66–80), (B) Lys⁷³ (residues 66–80), and (C) Lys⁷³ (residues 66–88). The bottom three panels are enlarged for the lower activities. The assays were performed at 37 °C and pH 8.0 for His⁷³ peptide using WT = 0.18 μm (20 min) and N255F/W273A = 0.72 μm (1 h) (A) or pH 10.5 for Lys⁷³ peptides using WT = 15 μm (3 h) and N255F/W273A = 0.18 μm (1 h) (B) or using WT = 3 μm (3 h) and N255F/W273A = 0.18 μm (1 h) (C). Data represent the mean ± S.D. of two independent determinations (n = 2) performed in duplicate.

Figure 3.

Structure of N255F/W273A in complex with Lys⁷³ in the active site. A, surface representation of the mutant enzyme (colored blue for positive, red for negative, and white for neutral charges) accommodation of peptide in a long surface groove. Omit electron density for ordered residues 66–82 (in stick model) is contoured at 3σ above the mean. B, Lys⁷³ is inserted into a narrow channel enveloped by Tyr³¹² and N255F and meets SAH in the bottom of the channel. C, the two mutated residues are separated in space. B and C, omit electron densities for Lys⁷³, N255F, and W273A, respectively, are contoured at 4σ above the mean. D, superimposition of SETD3 WT in complex with His⁷³ (PDB ID 6MBL) and N255F/W273A in complex with Lys⁷³ (PDB ID 6V62). Note the 5-bond distance (labeled 1–5 in red) between the main chain Cα atom and the target nitrogen. E and F, the distance between the sulfur atom and the target nitrogen atom is ∼3.4 Å. The water molecule w1 could facilitate the deprotonation of the target Nϵ-amino group of lysine during catalysis.

Figure 4.

Conserved active-site configuration for lysine methylation. A and B, SETD3 (N255F and W273A). C and D, LSMT. E and F, SETD6.

Figure 5.

SETD3 interaction with Gln⁷³. A, chemical structures of His, Lys, and Gln. The chemical bonds are numbered from the main chain Cα atom. B, SETD3 and the four mutants are not active on Gln⁷³ methylation. The assays were performed overnight at room temperature and pH 8.0 for His⁷³ or Gln⁷³ peptides using E = 3 μm, Peptide = 10 μm, and SAM = 40 μm. A 12% SDS-PAGE shows enzymes used in the assay. C, ITC K_D measurement. D, inhibition on His⁷³ methylation by Gln⁷³-containing peptide. The assays were performed at 37 °C and pH 8.0 using E = 0.18 μm, His⁷³ = 20 μm, and SAM = 40 μm. E, structure of SETD3 in complex with Gln⁷³-containing peptide (PDB ID 6V63). Omit electron density for Gln⁷³ is contoured at 4σ above the mean. F, a difference F_o − F_c electron density (in green) is contoured at 4σ above the mean. The 2F_o − F_c electron density (in gray) for the vicinity of the active site is contoured at 1.2σ above the mean. G, a model of SAM, with the transferable methyl occupies the green difference density. H, SETD3 is active on His⁷³ methylation without addition of SAM, indicating endogenous SAM was co-purified with SETD3. I, superimposition of Gln⁷³ and methyl-His⁷³ indicates the amide nitrogen atom of Gln⁷³ is not positioned in the primed location for catalysis. J, a model of Gln⁷³ clashes with Tyr³¹². Data represent the mean ± S.D. of two independent determinations (n = 2) performed in duplicate.