The DIM-5 protein (the smallest known member of the Suv39 family) contains four segments: a weakly conserved amino-terminal region (light blue), a pre-SET domain (yellow) containing nine invariant cysteines, the SET region (green) containing signature motifs NHXCXPN and ELXFDY (magenta), and the post-SET domain (gray) containing three invariant cysteines.

(A) Mass spectrometry analysis of different H3 peptides as DIM-5 substrates. The relative amount of each peptide species, expressed as a percentage of the sum of intensity of all related peaks, was plotted over the full time courses of the reactions.
(B) GRASP (Nicholls et al., 1991) surface charge distribution (blue for positive, red for negative, white for neutral). The H3 peptide and AdoHcy are shown as stick models.
(C) Ribbon (Carson, 1997) diagram colored as in Figure 1. The pre-SET residues (yellow) form a Zn₃Cys₉ triangular zinc cluster. The SET residues (green) and the N-terminal region are folded into six β sheets surrounding a knot-like structure (magenta). The post-SET residues (gray) bind the fourth zinc atom, adjacent to the substrate H3 peptide (red) and AdoHcy (blue).
(D) The substrate H3 peptide (red), superimposed on an omit electron density contoured at 4.0 σ (orange), is inserted as a parallel β strand (red in Figure 2C) between two DIM-5 strands, β 10 (green) and β 18 (magenta). The side chain density for H3 Arg-8 is complete at lower contour levels (2.5 σ in F_obs-F_cal and 0.8 σ in 2F_obs-F_cal).

(A) The post-SET zinc ion and the AdoHcy binding site. The zinc ion is presented as a red ball, coordinated by four cysteines, C244 (magenta) and C³⁰⁶XC³⁰⁸X₄C³¹³ (gray). AdoHcy is superimposed onto a difference electron density map contoured at 4.0 σ (orange). Dashed lines indicate the hydrogen bonds. One face of the AdoHcy adenine base lies against the aliphatic portion of R159, whose guanidino group forms a bifurcated salt bridge with the two carboxyl oxygen atoms of E278. The polar edge of the adenine base forms three hydrogen bonds to the DIM-5 backbone: the exocyclic amino group N6 with the carbonyl oxygen of H242, the ring N1 with the amide of L307, and the ring N7 with the amide of H242. The adenine ring carbon C8 makes van der Waals contacts to the Y283 hydroxyl and with the side chain carbonyl Oδ1 of N241; this explains the complete loss of AdoMet crosslinking in N241Q and Y283F mutants (Zhang et al., 2002). The two ribose hydroxyls interact with the main chain amide of V203 and the side chain carboxyl of D202. The amino group of the homocysteine moiety hydrogen bonds the side chain Oδ1 of N241, while its side chain amino group forms two hydrogen bonds with the backbone carbonyl of W161 and with the side chain of E278. The carboxylate group of the homocysteine moiety interacts with Y204 and the backbone amide of W161.
(B) Close-up view of the H3 peptide binding site with Lys9 inserted into a channel.
(C) The target Lys binding site (stereo). The arrow indicates the movement of the methyl group transferred from the AdoMet methylsulfonium group to the target amino group.
(D) DIM-5 activity (LogCPM) as a function of pH.
(E) AdoHcy bound in a large surface pocket, allowing for processive methylation. The green ellipse indicates the location where the AdoHcy homocysteine moiety binds in the peptide-free structure (Zhang et al., 2002).

(A) Activities of DIM-5 and SET7/9 mutants using histone substrate (top). AdoMet crosslinking experiments of DIM-5 showing fluorograph (middle) and Coomassie stain (bottom).
(B) Structure-based sequence alignment of DIM-5 and SET7/9. Secondary structures shown are based on Wilson et al. (2002) and Zhang et al. (2002). Vertical bars indicate residues that align spatially. Residues identical (black background) or similar (gray background) between the two enzymes, as well as the post-SET region of DIM-5, are highlighted. Numbered residues are described in the text. C-terminal hydrophobic residues of DIM-5 are underlined.
(C) Structural comparison of active sties in the ternary DIM-5 (in color) and binary SET7/9-AdoHcy (in black) (PDB 1MT6; Jacobs et al., 2002). The bound peptide in DIM-5 is represented as a solid electron density (orange), with the target Lys surrounded by either two Tyr and one Phe (DIM-5) or three Tyr (SET7/9).

(A) Representative spectra at various time points for WT DIM-5, its F281Y variant, WT SET7/9, and its Y305F variant are shown. The peaks for unmodified (Um) substrate and mono-, di-, and trimethylated products are labeled. Unlabeled minor peaks correspond to the sodium adducts of the major peaks (+23 Da).
(B) The relative amount of each peptide species over the full time courses of the reactions, expressed as a percentage of the sum of intensity of all related peaks.
(C) Spectra for three DIM-5 mutants having severely impaired catalytic activity but with normal product specificity.