Structural origins for the product specificity of SET domain protein methyltransferases Jean-Franc¸ois Couturea,1, Lynnette M. A. Dirkb, Joseph S. Brunzellec, Robert L. Houtzb, and Raymond C. Trievela,2 aDepartment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109; bDepartment of Horticulture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, KY 40546; and cDepartment of Molecular Pharmacology and Biological Chemistry, Life Sciences Collaborative Access Team, Northwestern University Center for Synchrotron Research, Argonne, IL 60439 Edited by David R. Davies, National Institutes of Health, Bethesda, MD, and approved October 30, 2008 (received for review July 11, 2008) SET domain protein lysine methyltransferases (PKMTs) regulate histone methyltransferases (Fig. S1). In lysine monomethyltrans- transcription and other cellular functions through site-specific ferases, this position is occupied by a tyrosine, whereas in most methylation of histones and other substrates. PKMTs catalyze the dimethyltransferases and trimethyltransferases, a phenylalanine formation of monomethylated, dimethylated, or trimethylated or another hydrophobic amino acid is located in this site (5–10). products, establishing an additional hierarchy with respect to These findings underpin a Phe/Tyr switch model for product methyllysine recognition in signaling. Biochemical studies of PK- specificity wherein the identity of the residue occupying the MTs have identified a conserved position within their active sites, switch position differentiates monomethyltransferases and di/ the Phe/Tyr switch, that governs their respective product specific- trimethyltransferases, with the exception of enzymes that are ities. To elucidate the mechanism underlying this switch, we have active within the context of heteromeric complexes whose sub- characterized a Phe/Tyr switch mutant of the histone H4 Lys-20 units regulate product specificity, such as ScSET1 (11) and (H4K20) methyltransferase SET8, which alters its specificity from a HsMLL (12, 13). Despite its biological importance, the molec- ular mechanism underlying the Phe/Tyr switch is poorly under- monomethyltransferase to a dimethyltransferase. The crystal stood. Indeed, this reflects a fundamental question: how are structures of the SET8 Y334F mutant bound to histone H4 peptides methyl groups of monomethyllysine, dimethyllysine, and trimeth- bearing unmodified, monomethyl, and dimethyl Lys-20 reveal that yllysine physically arranged in the active sites of PKMTs during the phenylalanine substitution attenuates hydrogen bonding to a successive rounds of methylation? This question has remained structurally conserved water molecule adjacent to the Phe/Tyr unaddressed because of a lack of structures of SET domain PKMTs switch, facilitating its dissociation. The additional space generated bound to lysine substrates in different methylation states. by the solvent’s dissociation enables the monomethyllysyl side To resolve these issues and gain mechanistic insights into SET chain to adopt a conformation that is catalytically competent for domain product specificity, we have structurally and functionally dimethylation and furnishes sufficient volume to accommodate characterized a Phe/Tyr switch mutant of the H4K20-specific the dimethyl ␧-ammonium product. Collectively, these results in- monomethyltransferase SET8 (also known as PR-SET7 and dicate that the Phe/Tyr switch regulates product specificity KMT5A). We previously reported that a phenylalanine mutation through altering the affinity of an active-site water molecule of the Phe/Tyr switch residue Tyr-334 alters the product spec- whose dissociation is required for lysine multiple methylation. ificity of SET8 to a dimethyltransferase, but does not affect BIOCHEMISTRY histone H4 binding or the overall methylation rate (6). We chose enzyme mechanism ͉ histone methylation ͉ transcription ͉ chromatin ͉ this mutant for further analysis to evaluate how a subtle mutation enzyme kinetics from tyrosine to phenylalanine can fundamentally alter product specificity. Crystal structures of SET8 Y334F in complex with a histone H4 peptide bearing an unmodified (H4K20), monom- ysine methylation is a widespread covalent modification that ethylated (H4K20me1), and dimethylated Lys-20 (H4K20me2) Loccurs in histones and nonhistone proteins. Site-specific reveal that the Phe/Tyr switch modulates the affinity of a methylation of lysines in histones H3, H4, and H1b has been structurally conserved water molecule within the active site. implicated in a myriad of functions, including transcriptional Dissociation of this water molecule permits a monomethyllysine regulation, heterochromatin formation, and DNA damage re- substrate to adopt an alternative conformation conducive for sponse (1). Various effector proteins mediate these functions dimethylation. Together, these findings demonstrate that the through sequence-specific recognition of lysine methyl marks. In Phe/Tyr switch governs SET domain product specificity through addition, the lysine ␧-amine group can be monomethylated, the intermediacy of a water molecule. dimethylated, or trimethylated, imparting a further level of specificity in methyllysine signaling. The importance of this Results specificity has been emphasized by recent studies illustrating that Kinetic Characterization of SET8 Y334F Mutant. In our initial studies, specific methylation states are enriched in distinct regions of we reported the kinetic parameters of the SET8 Y334F mutant genes (2). Correlatively, certain methyllysine binding domains discriminate among different degrees of lysine methylation (3), underscoring the synergy between the site and degree of meth- Author contributions: R.L.H. and R.C.T. designed research; J.-F.C., L.M.A.D., and J.S.B. performed research; L.M.A.D. and R.L.H. contributed new reagents/analytic tools; J.-F.C., ylation in signaling. L.M.A.D., J.S.B., R.L.H., and R.C.T. analyzed data; and J.-F.C., L.M.A.D., R.L.H., and R.C.T. A key to deciphering the functions of lysine methylation is to wrote the paper. understand the specificities of the enzymes that establish these The authors declare no conflict of interest. marks. Structural studies of PKMTs belonging to the SET This article is a PNAS Direct Submission. domain family have yielded insights into the mechanisms by Data deposition: The atomic coordinates and structure factors have been deposited in the which these enzymes recognize and methylate specific sites Protein Data Bank, www.pdb.org (PDB ID codes 3F9W, 3F9X, 3F9Y, and 3F9Z). within their cognate substrates (4). Protein substrates generally 1Present address: Ottawa Institute of Systems Biology, Department of Biochemistry, Mi- bind in a ␤-strand conformation, depositing the lysyl side chain crobiology, and Immunology, University of Ottawa, Ottawa, ON, Canada K1H 8M5. in a channel that traverses the core of the catalytic domain and 2To whom correspondence should be addressed. E-mail: [email protected]. links to the S-adenosyl-L-methionine (AdoMet) binding site. This article contains supporting information online at www.pnas.org/cgi/content/full/ Mutational analyses have revealed a specific site within the lysine 0806712105/DCSupplemental. binding channel that modulates the product specificities of © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806712105 PNAS ͉ December 30, 2008 ͉ vol. 105 ͉ no. 52 ͉ 20659–20664 Downloaded by guest on September 23, 2021 Table 1. Biochemical analysis of native SET8 and the Y334F mutant Ϫ1 Ϫ1 Ϫ1 3 SET8 Peptide substrate KD,␮M KM, ␮M kcat, min kcat/KM, ␮M ϫ min ϫ 10 Native H4K20* 33 Ϯ 1 400 Ϯ 30 0.43 Ϯ 0.02 1.1 Ϯ 0.1 H4K20me1 24 Ϯ 1NANA NA Y334F† H4K20 19 Ϯ 2* 490 Ϯ 40 0.43 Ϯ 0.09 0.88 Ϯ 0.20 H4K20me1 32 Ϯ 1 260 Ϯ 30 0.016 Ϯ 0.001 0.062 Ϯ 0.008 NA, no activity detected. *Data previously reported and shown for comparative purposes (6). [Reproduced with permission from ref. 6 (Copyright 2005, Cold Spring Harbor Lab Press).] †No heat of binding was detected between SET8 Y334F and the H4K20me2 peptide by isothermal titration calorimetry (ITC). Error values were calculated from the nonlinear fits to the kinetic and ITC data. (residues 191–352) by using a 10-residue H4K20 peptide sub- rimmed methyltransfer pore through which a methyl group Ϫ1 strate (KM ϭ 520 ␮M, kcat ϭ 0.45 min ), representing the transits during catalysis (15). In both native SET8 and the Y334F aggregate rates of monomethylation and dimethylation (6). To mutant, the conformation of the K20 side chain is oriented via measure the individual rate constants for these reactions, SET8 hydrogen bonding to the Tyr-245 OH group and a tightly bound Y334F was assayed with an H4K20me1 peptide and a H4K20 water molecule that occupies a cleft in the active site that we peptide by using short assay times that exclude the formation of refer to as the solvent pocket. In the native enzyme, the water the dimethylated product. Under these conditions, SET8 Y334F molecule is further coordinated within this pocket via hydrogen displays comparable KM values for the 2 peptides and similar bonds to the Tyr-334 OH group and the carbonyl oxygens of binding affinities, as measured by isothermal titration calorim- Gly-294 and Ile-297 (Fig. 2A), whereas in SET8 Y334F, the etry, but methylates the H4K20 peptide more efficiently than Tyr-334 hydrogen bond to the water is absent because of the H4K20me1 (Table 1). The k value for the H4K20me1 peptide cat phenylalanine mutation (Fig. 2B).