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Raymond C Trievel

Publications and source records attributed to Raymond C Trievel.

10 recordsLinked to original sources
PubMed2006

Histone-modifying enzymes: encrypting an enigmatic epigenetic code.

Histone-modifying enzymes catalyze a diverse array of post-translational modifications of core and linker histones within chromatin. These modifications govern a multitude of genomic functions, particularly gene expression, and are believed to constitute an epigenetic code. Histone-modifying enzymes inscribe this code by catalyzing site-selective modifications, which are subsequently interpreted by effector proteins that recognize specific covalent marks. The substrate specificity of these enzymes is of fundamental biological importance because it underpins this epigenetic code. Recently, the structural basis of this specificity has been examined with regards to recently determined structures of GCN5 acetyltransferases and SET domain methyltransferases in complex with their cognate histone substrates.

Animals↗
PubMed2006

Molecular recognition of histone H3 by the WD40 protein WDR5.

The WD40-repeat protein WDR5 is a conserved subunit of Trithorax (TRX) histone methyltransferase complexes. WDR5 has been reported to selectively bind dimethylated Lys4 (K4me2) in histone H3 to promote K4 trimethylation by TRX. To elucidate the basis of this binding specificity, we have determined the crystal structure of WDR5 bound to a histone H3 peptide bearing K4me2. The structure reveals that the N terminus of histone H3 binds as a 3(10)-helix in the central depression formed by the WD40 repeats. R2 in histone H3 is bound in the acidic channel in the protein's core, whereas K4me2 is solvent exposed and does not engage in direct interactions with WDR5. Functional studies confirm that WDR5 recognizes A1, R2 and T3 in histone H3 but has virtually identical affinities for the unmodified and mono-, di- and trimethylated forms of K4, demonstrating that it does not discriminate among different degrees of methylation of this residue.

Amino Acid Sequence↗
PubMed2006

Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases.

SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine epsilon-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product epsilon-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the epsilon-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine epsilon-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.

Arabidopsis↗
PubMed2006

Structural basis for the methylation site specificity of SET7/9.

Human SET7/9 is a protein lysine methyltransferase (PKMT) that methylates histone H3, the tumor suppressor p53 and the TBP-associated factor TAF10. To elucidate the determinants of its substrate specificity, we have solved the enzyme's structure bound to a TAF10 peptide and examined its ability to methylate histone H3, TAF10 and p53 substrates bearing either mutations or covalent modifications within their respective methylation sites. Collectively, our data reveal that SET7/9 recognizes a conserved K/R-S/T/A motif preceding the lysine substrate and has a propensity to bind aspartates and asparagines on the C-terminal side of the lysine target. We then used a sequence-based approach with this motif to identify novel substrates for this PKMT. Among the putative targets is TAF7, which is methylated at Lys5 by the enzyme in vitro. These results demonstrate the predictive value of the consensus motif in identifying novel substrates for SET7/9.

Amino Acid Sequence↗
PubMed2005

A coupled fluorescent assay for histone methyltransferases.

Histone methyltransferases (HMTs) catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of lysines and arginines in the nucleosomal core histones H3 and H4 and the linker histone H1b. Methylation of these residues regulates either transcriptional activation or silencing, depending on the residue modified and its degree of methylation. Despite an intense interest in elucidating the functions of HMTs in transcriptional regulation, these enzymes have remained challenging to quantitatively assay. To characterize the substrate specificity of HMTs, we have developed a coupled-fluorescence-based assay for AdoMet-dependent methyltransferases. This assay utilizes S-adenosylhomocysteine hydrolase (SAHH) to hydrolyze the methyltransfer product S-adenosylhomocysteine (AdoHcy) to homocysteine (Hcy) and adenosine (Ado). The Hcy concentration is then determined through conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore. Using this assay, we have determined the kinetic parameters for the methylation of a synthetic histone H3 peptide (corresponding to residues 1-15 of the native protein) by Schizosaccharomyces pombe CLR4, an H3 Lys-9-specific methyltransferase. The fluorescent SAHH-coupled assay allows rapid and facile determination of HMT kinetics and can be adapted to measure the enzymatic activity of a wide variety of AdoMet-dependent methyltransferases.

Adenosylhomocysteinase↗
PubMed2005

Structural and functional analysis of SET8, a histone H4 Lys-20 methyltransferase.

SET8 (also known as PR-SET7) is a histone H4-Lys-20-specific methyltransferase that is implicated in cell-cycle-dependent transcriptional silencing and mitotic regulation in metazoans. Herein we report the crystal structure of human SET8 (hSET8) bound to a histone H4 peptide bearing Lys-20 and the product cofactor S-adenosylhomocysteine. Histone H4 intercalates in the substrate-binding cleft as an extended parallel beta-strand. Residues preceding Lys-20 in H4 engage in an extensive array of salt bridge, hydrogen bond, and van der Waals interactions with hSET8, while the C-terminal residues bind through predominantly hydrophobic interactions. Mutational analysis of both the substrate-binding cleft and histone H4 reveals that interactions with residues in the N and C termini of the H4 peptide are critical for conferring substrate specificity. Finally, analysis of the product specificity indicates that hSET8 is a monomethylase, consistent with its role in the maintenance of Lys-20 monomethylation during cell division.

Binding Sites↗
PubMed2004

Structure and function of histone methyltransferases.

Histones are the major protein constituent of chromatin in the eukaryotic nucleus. These proteins undergo a host of different post-translational modifications, including phosphorylation, acetylation, and methylation, which have profound effects on the remodeling of chromatin. Histone modifications can function either individually or combinatorially to govern such processes as transcription, replication, DNA repair, and apoptosis. Recent studies have focused on histone arginine and lysine methylation and the roles of these modifications in transcriptional regulation and the establishment of heterochromatin. Concomitantly, several families of histone methyltransferases (HMTs) have been identified that catalyze the methylation of specific arginines or lysines in histones H3 and H4. Not surprisingly, many of these methyltransferase genes had been previously identified as important genetic regulators in organisms such as yeast and Drosophila, which underscores the importance of histone methylation in transcriptional control and chromatin remodeling. Structures of several representatives of these HMT families have recently been determined, yielding insight into their catalytic mechanism and histone substrate specificity. The focus of this review is to briefly summarize the roles of histone methylation in chromatin remodeling and to discuss the structures, substrate specificities, and mechanisms of the different classes of HMTs.

Animals↗
PubMed2003

Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT.

SET domain protein methyltransferases catalyze the transfer of methyl groups from the cofactor S-adenosylmethionine (AdoMet) to specific lysine residues of protein substrates, such as the N-terminal tails of histones H3 and H4 and the large subunit of the Rubisco holoenzyme complex. The crystal structures of pea Rubisco large subunit methyltransferase (LSMT) in ternary complexes with either lysine or epsilon-N-methyllysine (MeLys) and the product S-adenosylhomocysteine (AdoHcy) were determined to resolutions of 2.65 and 2.55 A, respectively. The zeta-methyl group of MeLys is bound to the enzyme via carbon-oxygen hydrogen bonds that play a key role in catalysis. The methyl donor and acceptor are aligned in a linear geometry for S(N)2 nucleophilic transfer of the methyl group during catalysis. Differences in hydrogen bonding between the MeLys epsilon-amino group and Rubisco LSMT and SET7/9 explain why Rubisco LSMT generates multiply methylated Lys, wheras SET7/9 generates only MeLys.

Amino Acid Sequence↗
PubMed2002

Structure and catalytic mechanism of a SET domain protein methyltransferase.

Protein lysine methylation by SET domain enzymes regulates chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. The 2.6 A resolution structure of Rubisco large subunit methyltransferase in a pseudo-bisubstrate complex with S-adenosylhomocysteine and a HEPES ion reveals an all-beta architecture for the SET domain embedded within a larger alpha-helical enzyme fold. Conserved regions of the SET domain bind S-adenosylmethionine and substrate lysine at two sites connected by a pore. We propose that methyl transfer is catalyzed by a conserved Tyr at a narrow pore connecting the sites. The cofactor enters by a "back door" on the opposite side of the enzyme from substrate, promoting highly specific protein recognition and allowing addition of multiple methyl groups.

Amino Acid Motifs↗