Crystal structures of human CDY proteins reveal a crotonase‐like fold

The human chromodomain Y-related (CDY) protein family contains six proteins (CDY1, CDY1B, CDY2A, CDY2B, CDYL, and CDYL2).1, 2 The four closely related genes (CDY1, CDY1B, CDY2A, and CDY2B) encoding these proteins are located on chromosome Y and the resultant proteins exhibit more than 96% sequence identity. The other two members, CDYL (chromodomain protein, Y-like) and CDYL2 genes are located on autosomes. CDYL and CDYL2 proteins only share 43% sequence identity. The sequence identity between the proteins encoded by autosomal genes and chromosomal genes is also lower than that observed among the proteins encoded by the four chromosomal genes. A further distinction between the autosomal genes and their chromosomal counterparts is that the chromosomal genes are only present in primates, whereas the CDYL and CDYL2 genes exist in most mammalian species.2-6 It is believed that the progenitor of the CDY gene family arose de novo in the mammalian ancestor via domain accretion. This progenitor later duplicated to generate the two autosomal genes CDYL and CDYL2. Before human-mouse divergence, a processed CDYL mRNA retroposed onto the Y chromosome to create the CDY gene,2, 6, 7 which subsequently amplified into multiple copies on the chromosome. In humans, the autosomal CDYL genes are ubiquitously expressed, whereas the CDY genes are only expressed in testis, thus implicating the CDY family of proteins functions in both somatic development and spermatogenesis.2 In mammals, spermatogenesis occurs in the male testes and epididymis in a stepwise fashion and is essential for sexual reproduction.8 The spermatogenic function requires dramatic chromatin remodeling caused by hyperacetylation of histones during spermatid maturation,1, 2 but the histone acetyltransferase responsible for this modification has not yet been identified. The correlation between deletion of CDY genes on Y chromosome in human and spermatogenic failure suggested the role of CDYs in spermatogenesis.1 However, the mechanism by which CDYs interfere with spermatognesis remains unclear. The CDY family of proteins contain two functional domains: a chromo domain involved in chromatin binding and a catalytic domain found in many Coenzyme A (CoA)-dependent acylation enzymes1, 2, 9 (Fig. 1). Similar domain architectures have also been found in other histone acetyltransferases such as the MYST family of histone acetyltransferases.10 The presence of these two domains in CDYs suggests a role for this protein family in histone modification and recognition. The chromo domain of human CDY has been shown to interact with methylated lysine 9 of histone H3.11, 12 The catalytic domain, however, has been implicated in different chromatin modification process. Biochemical studies showed that CDYs possess in vitro histone acetyltransferase activity.13 The recombinant proteins were able to acetylate histones H4 and H2A. Meanwhile, this domain is also found to recruit histone deacetylases to sites within somatic cells.14 Schematic illustration of domain organization of CDY family. The chromodomain and catalytic domains are colored in red and blue. The boundaries of each domain are labeled. The sequences used to generate the figure are as follows: CDY1, NP_004671; CDY1B, NP_001003895; CDY2A, NP_004816; CDY2B, NP_001001722; CDYL, NP_004815; CDYL2, NP_689555. Due to the different chromatin modification processes CDYs are involved in13, 14 and the functional importance of spermatogenesis, we aimed to solve the structures of human CDYs and compare their structural properties to further understand the functional role of CDYs in spermatogenesis. Here, we report the crystal structures of the catalytic domain of human CDY1, CDY2B, and CDYL, at 2.28, 2.20, and 1.97 Å resolutions, respectively. The structures did not show similarity to any known structure of histone acetyltransferases. Instead, they all exhibit a three-dimensional structure most similar to the crotonase superfamily of proteins. AcCoA, acetyl-coenzyme A; BCA, 4-hydroxybenzoyl coenzyme A; CarB, carboxymethylproline synthase; CDY, chromodomain protein, Y-related; 4CBD, 4-chlorobenzoyl coenzyme A dehalogenase; ECH, enoyl-CoA hydratase; ECI,Δ3,Δ2-enoyl CoA isomerase; RMSD, root-mean-square deviation. DNA fragments encoding the catalytic domains of human CDY proteins were amplified by PCR and subcloned into pET28a-LIC vector, downstream of the poly-histidine coding region. The CDY proteins were overexpressed in E. coli BL21 (DE3) codon plus RIL strain (Stratagene) by addition of 1 mM isopropyl-1-thio-D-galactopyranoside and incubated overnight at 15°C. Harvested cells were resuspended in 50 mM HEPES buffer, pH 7.4, supplemented with 500 mM NaCl, 5 mM imidazole, 2 mM β-mercaptoethanol, 5% glycerol. The cells were lysed by passage through a microfluidizer (Microfluidics Corporation) at 20,000 psi and the supernatants were clarified via centrifugation. The soluble lysate was loaded onto a 5 mL HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 column volumes of 20 mM HEPES buffer, pH 7.4, containing 500 mM NaCl, 50 mM imidazole, and 5% glycerol, and the protein was eluted with 20 mM HEPES buffer, pH 7.4, 500 mM NaCl, 250 mM imidazole, 5% glycerol. The eluted proteins were dialyzed against buffer containing 20 mM HEPES buffer, pH 7.4, 500 mM NaCl, 5% glycerol, and further purified to homogeneity by ion-exchange chromatography. The purified CDY proteins (10 mg/mL) were crystallized using the hanging drop vapor diffusion method at 20°C by mixing equal volume of the protein solution with the reservoir solution. For CDY1, the protein was crystallized in 2.0M ammonium sulfate, 0.2M K/Na tartrate, 0.1M Bis-Tris, pH 5.5; CDY2B was crystallized in 8% PEG 4,000, 0.1M sodium acetate pH 4.6; CDYL was crystallized in 12% iso-propanol, 0.2M sodium citrate, 0.1M sodium cacodylate, pH 5.0. The crystals were frozen in liquid nitrogen in mother liquor supplemented with 20% glycerol as cryoprotectant. X-ray diffraction data were collected at 100 K at beamline 17ID of Advanced Photon Source (APS) at Argonne National Laboratory and in-house Rigaku FR-E. Data were processed using the HKL-2000 software suite.15 The structure of CDY1 was solved by single-wavelength anomalous diffraction (SAD) using a selenium methionine derivative crystal with the program SHELXD,16 and the phasing was performed using SHELXE.17 The structure of CDY1 (PDB code 2FBM) was then used as model to solve CDY2B and CDYL structures by molecular replacement using MOLREP.18 ARP/wARP19 was used for automatic model building. The graphics program COOT20 was used for model building and visualization. Refmac21 was employed for refinement of the structures. Crystal diffraction data and refinement statistics for the structures are displayed in Table I. We have determined the crystal structures of CDY1, CDY2B, and CDYL and refined them to 2.28, 2.20, and 1.97 Å resolution, respectively, with excellent refinement statistics (Table I). The three-dimensional structures of CDY1, CDY2B, and CDYL exhibit a high degree of structural similarity, despite difference in crystallographic packing (Fig. 2). The crystals of CDY1 and CDYL both contain three protein molecules per crystallographic asymmetric unit, and the three molecules are arranged in the shape of a propeller [Fig. 2(B)]. The CDY2B crystal contains six protein molecules per crystallographic asymmetric unit, which form a dimer of trimers. The trimers also exhibit good structural similarity to the trimers of CDY1 and CDYL that result in interactions between neighboring asymmetric units of the respective crystals. For each protein, the monomers are virtually superimposable. As an example, in CDYL, the three chains can be aligned with a root-mean-square deviation (RMSD) of 0.258–0.433 Å for all Cα atoms. Overall structure of CDY family of proteins. (A) Cartoon representation of CDYL monomer. The secondary structure elements are labeled. (B) Cartoon representation of trimeric structures of CDYL, CDY1 and CDY2B. (C) Oligomerization interface of CDYL. The interface between chain A and B is shown. The two chains are shown in cartoon and surface representation and colored in yellow and green. The secondary structure elements and amino acids involved in the interface formation are labeled. Similar interfaces are also observed between chain A and C, chain B and C. Figures are generated by using PyMOL (DeLano Scientific, Palo Alto, CA). Trimeric association of the CDYL monomers results from the formation of extensive interfaces between the subunits. In CDYL structure, about 17% of the total solvent accessible surface of each monomer is buried through trimerization, with the interface between chain A and B slightly larger (1062.7 Å2) than the ones formed between chain B and C (1026.3 Å2) and chain A and C (1020.8 Å2). For each interface, the main contact region involves the C-terminus of each monomer. Helix α9 of chain A protrudes into the complementary pocket formed by helices α5, α6, α7, and α10 of chain B. In addition, strands β8 and β11 of chain A also make contacts with chain B [Fig. 2(C)]. The side chains of W127 and E213 from chain A form hydrogen bonds with the side chains of E164 and K239 from chain B. Residue S184 of chain A establishes three hydrogen bonds with residues from chain B: the side chain of S184 hydrogen bonds with carbonyl oxygen of A160, whereas the carbonyl oxygen of S184 interacts with the side chains of R170 and K180. Beside these hydrogen bonds, a network of hydrophobic interactions is also established between chain A and B. These interactions involve residues D124, Q185, V210, L217, and V218 from chain A, and residues Q143, T150, P154, N163, L166, R232, and V236 from chain B. In CDY1 and CDY2B structures, the three monomers have the same organization as seen above, with an average 16–17% of each monomer surface buried by trimerization. In CDY2B crystals, the two trimers are packed to form a hexamer, although there are few contacts between the trimers. The overall structures of each monomer of CDY2B, CDY1, and CDYL are very similar (Fig. 2). The Cα atoms of 258 structurally equivalent residues of CDY2B and CDYL can be superimposed with a RMSD of 0.737 Å, whereas CDY1 and CDYL, CDY1 and CDY2B can be superimposed with a RMSD of 0.619 Å and 0.355 Å, respectively. The overall structure of CDY proteins has a α/β fold. The N-terminal portion of each chain contains a core of 4 β-β-α structure elements, flanked by two β-strands (β1 and β11). A α-β-α structure element (α6-β9-α7) precedes strand β11. The strands in the structure are mainly parallel, except strand β1, which is in antiparallel orientation with strand β2. The strands form two β-sheets: one contains strands β1, β2, β4, β6, β8, and β11; the second consists of strands β3, β5, β7, β9, and β10. These two sheets are in almost perpendicular orientation to each other. The C-terminal structure is mainly helical, consisting of helices α8-α11. The four helices wraps around the core β sheet pair [Fig. 2(A)]. CDY protein was previously characterized as histone acetyltransferases in vitro.13 However, our structures of the CDY family proteins did not show structural similarity to any known histone acetyltransferase structures. To further explore the function of CDY proteins, we compared the three structures of the CDY proteins against a non-redundant set of structures. DALI22 search revealed a set of closely related structures to CDY proteins but none of these proteins is histone acetyltransferase. Instead, the core fold of CDY proteins showed strong similarity to the fold of crotonase-like superfamily (Fig. 3). Due to high structure similarity within CDY family, we used CDYL as an example to show the comparison results (Table II). Comparison of CDYL and 4CBD structures. (A) The structures of CDYL monomer and 4CBD monomer (protein data bank code: 1NZY) are shown in cartoon representation. The bound ligand 4-hydroxybenzoyl coenzyme A for 4CBD is shown in stick-and-ball. The superimposition of the two structures is on the right side. (B) Close-up stereo view of the active site. The critical residues for the reaction in 4CBD and their structural equivalents in CDYL are shown in stick-and-ball representation, with the carbon atoms in yellow and cyan, respectively. (C) Multiple sequence alignment of representative members of CDY family and crotonase superfamily. Identical residues are colored in red in the alignment. Secondary structure elements of human CDYL are assigned by PROCHECK program23 and shown above the sequences: the helices are shown as cylinders and strands are shown as arrows. The residues in 4CBD that interact with the ligand BCA are labeled by stars under the sequence. The alignment was generated using Clustal W24 assisted with hand fittings. The sequences shown are as follows: human CDYL (CDYL, NP_004815), human CDY2B (CDY2B, NP_001001722), human peroxisomal 3,2-trans-enoyl-CoA isomerase (PECI, NP_006108), Erwinia carotovora carboxymethylproline synthase CarB (CarB, AAD38230), and Pseudomonas sp. 4-chlorobenzoyl coenzyme A dehalogenase (4CBD, ABQ44578). Structurally, the crotonase superfamily members can be divided into three groups, based on the position of their C-terminal helices.25 The first group, including enoyl-CoA hydratase (ECH) and 4-chlorobenzoyl-CoA dehalogenase (4CBD),26 has a C-terminus that protrudes away from the core of its monomer and covers the active site of an adjacent monomer of the same trimer. In the second group of proteins, including Δ3,Δ2-enoyl-CoA isomerase (ECI),27 the C-terminus folds back over the core to cover the active site. The third group, including 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB), has a C-terminus crosses the trimer–trimer interface and forms part of the active site of a monomer from another (opposing) trimer.28 Based on the position of the C-terminus, human CDY proteins belong to the second group, since in all three structures, the C-terminus of one monomer does not crossover to another monomer or another trimer, but folds back over the active site where it is likely involved in substrate binding [Fig. 3(A)]. Most of the available structures of crotonase superfamily members are apo structures, likely due to the tight crystal packing. Among the structurally similar proteins to CDYs, there are two complex structures available: 4-chlorobenzoyl-CoA dehalogenase (4CBD) in complex with 4-hydroxybenzoyl-coenzyme A (BCA) (PDB code 1NZY)26 and carboxymethylproline synthase CarB in complex with acetyl-coenzyme A (AcCoA) (PDB code: 2A81).25 A comparison of CDY structures with these two complex structures revealed that the cofactor-binding site of CDYs can be roughly mapped to the relative open cavity between the second β-sheet (strands β3, β5, β7, β9, and β10) and the C-terminus of each monomer [Fig. 3(A)]. Due to the protruding C-terminus, the cofactor binding site of 4CBD also involves several residues from the adjacent subunit. As in CDY proteins, this interaction would most likely come from the C-terminus of the same subunit [Fig. 3(C)]. Crotonase superfamily contains a group of divergently related enzymes that catalyze a wide range of metabolic reactions employing different mechanisms.29 Three-dimensional structure and amino acid sequence comparison of the crotonase superfamily showed that no active site catalytic groups are strictly conserved within this superfamily. However, these enzymes seem to require a common feature of stabilizing of an enolate anion intermediate derived from an acyl-CoA substrate.29 The presence of an “oxyanion hole”29, 30 is the hallmark of this family of enzymes. To identify the critical residues for formation of the oxyanion hole in CDYL, we superimposed the structure of 4CBD and CDYL [Fig. 3(B)]. In 4CBD, the oxyanion hole involves the amide nitrogens of residues F64 and G114. The structural equivalents of these two residues in CDYL are L403 and L452. The amide nitrogens of these two residues superimpose very well with those in F64 and G114 of 4CBD. Residue G114 (and its equivalents in crotonase superfamily) usually is located at the N-terminus of a α-helix, as the helix dipole moment may play a role in stabilizing the intermediate during the reaction.29 In CDYL, L403 is at the N-terminus of helix α4. At the active site of crotonases, an aspartic acid or a glutamic acid is also involved in proton transfer. In 4CBD, this function is accomplished by D145, whereas in CDYL, the structurally corresponding residue is D483. Similar residues are also observed in the putative active site of CDY2B and CDY1. The analysis of the active site of CDY proteins indicates that this family of proteins has the residues that are required for the function of crotonase family of proteins. Previous studies provided evidence linking CDY family of proteins with hyperacetylation during spermiogenesis.13 We carried out structural studies on CDY family of proteins to gain better understanding of the functional role of CDYs in spermatogenesis. Our structures of CDY proteins did not show similarity to known histone acetyltransferase structures. The CDY family of proteins has a similar α/β structure fold with that of the crotonase superfamily. The comparative analysis of the active site of CDYL and 4CBD revealed the presence of critical residues for crotonase superfamily catalytic activity in CDYL. Even though our structural analysis revealed some conserved features of the active site, the detailed function and reaction mechanism for CDY proteins remain unsolved. The role of CDY family in spermiogenesis also remains unknown after uncover of crotonase structure in this family. However, our observations of the structural relationship of CDYs to several enzymes that bind to acetyl-CoA-like cofactors suggest the possibility of AcCoA-binding for CDYs. It is still possible for CDYs to utilize the crotonase fold for CoA-dependent HAT activity. Further biochemical and cell biological studies are essential to address these questions. The authors thank Peter Loppnau for his excellent technical support and Dr. Melanie Adams-Cioaba for critical reading of the manuscript. The SGC is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co. Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust. Data for this study were measured at beamline 17ID of Advanced Photon Source (APS) at Argonne National Laboratory.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.