Biomedical subjects

Jean-François Couture

Publications and source records attributed to Jean-François Couture.

9 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↗

PubMed2005

Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen.

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗

PubMed2004

Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily.

The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids). In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.08A resolution, respectively. Inspection of the cofactor-binding cavity led to the identification of a new interaction between side-chains of residues His222 and Lys270, which cover the central phosphate chain of the cofactor, reminiscent of the "safety-belt" found in other aldo-keto reductases. Testosterone is stabilized by a phenol/benzene tunnel composed of side-chains of numerous residues, among which Phe54, which forces the steroid to take up an orientation markedly contrasting with that found in HSD ternary complexes reported. Combining structural, site-directed mutagenesis, kinetic and fluorescence titration studies, we found that the selectivity of rb20alpha-HSD is mediated by (i) the relaxation of loop B (residues 223-230), partly controlled by the nature of residue 230, (ii) the nature of the residue found at position 54, and (iii) the residues found in the C-terminal tail of the protein especially the side-chain of the amino acid 306.

20-alpha-Hydroxysteroid Dehydrogenase↗

PubMed2003

Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids.

Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.59A resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the N(epsilon ) atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3alpha-HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H(222)I substitution decreases the K(m) value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R(304)L substitution leads to a 65-fold decrease in the K(m) value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.

20-Hydroxysteroid Dehydrogenases↗