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Structural origins for the product specificity of SET domain protein methyltransferases - PubMed

  • ️Tue Jan 01 2008

Structural origins for the product specificity of SET domain protein methyltransferases

Jean-François Couture et al. Proc Natl Acad Sci U S A. 2008.

Abstract

SET domain protein lysine methyltransferases (PKMTs) regulate transcription and other cellular functions through site-specific methylation of histones and other substrates. PKMTs catalyze the formation of monomethylated, dimethylated, or trimethylated products, establishing an additional hierarchy with respect to methyllysine recognition in signaling. Biochemical studies of PKMTs have identified a conserved position within their active sites, the Phe/Tyr switch, that governs their respective product specificities. To elucidate the mechanism underlying this switch, we have characterized a Phe/Tyr switch mutant of the histone H4 Lys-20 (H4K20) methyltransferase SET8, which alters its specificity from a monomethyltransferase to a dimethyltransferase. The crystal structures of the SET8 Y334F mutant bound to histone H4 peptides bearing unmodified, monomethyl, and dimethyl Lys-20 reveal that the phenylalanine substitution attenuates hydrogen bonding to a structurally conserved water molecule adjacent to the Phe/Tyr switch, facilitating its dissociation. The additional space generated by the solvent's dissociation enables the monomethyllysyl side chain to adopt a conformation that is catalytically competent for dimethylation and furnishes sufficient volume to accommodate the dimethyl epsilon-ammonium product. Collectively, these results indicate that the Phe/Tyr switch regulates product specificity through altering the affinity of an active-site water molecule whose dissociation is required for lysine multiple methylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Analysis of the product specificities of native SET8 and the Y334F mutant. TLC-based assays of SET8 and the Y334F mutant with the unmodified H4K20 peptide and 3H-AdoMet as substrates are illustrated. The monomethyllysine and dimethyllysine products generated by native SET8 and the Y334F mutant were separated on a TLC plate (Lower), extracted, quantified, and displayed as a bar graph (Upper).

Fig. 2.
Fig. 2.

Structures of the active sites of native SET8 and the Y334F mutant bound to AdoHcy and H4K20, H4K20me1, and H4K20me2 peptides. His-18 in histone H4 forms 1 wall of the channel and was omitted to provide an unobstructed view of the active site. SET8, histone H4, and AdoHcy are delineated by gray, gold, and green carbon atoms, respectively. The Y334F mutation is highlighted in magenta. (Insets) Shown are the FO − FC omit map electron densities for the various K20 side chains contoured at 2.0 σ. Conventional and CH…O hydrogen bonds are depicted as orange and cyan dashes, respectively. (A) Native SET8–H4K20–AdoHcy complex (Protein Data Bank ID code 1ZKK). (B) SET8 Y334F–H4K20–AdoHcy. (C) SET8 Y334F–H4K20me1–AdoHcy. (D) SET8 Y334F–H4K20me2–AdoHcy.

Fig. 3.
Fig. 3.

Monomethyllysine and dimethyllysine binding by SET8 Y334F. H4K20me1 (green carbons) and H4K20me2 (orange carbons) are superimposed within the active site. Hydrogen bonds between the active site and the K20me1 and K20me2 side chains are illustrated according to the corresponding colors of the carbon atoms in their side chains, and hydrogen bonds common to both complexes are shown in violet.

Fig. 4.
Fig. 4.

Model for H4K20 dimethylation by the SET8 Y334F mutant. (A) First methyltransfer reaction. In the SET8 Y334F–H4K20–AdoMet complex, AdoMet was modeled in the active site by using the AdoHcy coordinates (Fig. 2 color scheme with hydrogens rendered in cyan). The SN2 reaction distance and bond angle corresponding to the H4K20 ε-amine group and the AdoMet methyl group and sulfonium cation are noted.

Fig. S6

illustrates the hydrogen bonding to the K20 ε-amine. (B) Second methyltransfer reaction. In this substrate complex, the H4K20me1 side chain is modeled with its methyl group projecting into the vacated water binding site, which is inferred from the coordinates of the corresponding methyl group in the H4K20me2 product complex (Fig. 2D). This orientation aligns the K20me1 ε-amine for methyltransfer with AdoMet.

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