Chromodomain蛋白在表观遗传调控中的功能

克罗莫结构域 (chromatin organization modifier domain, chromodomain)是与染色质结构相关的进化上保守的蛋白质模体。Chromodomain中芳香族氨基酸残基组成保守的疏水“box”结构与“组蛋白密码”中的二甲基或三甲基修饰的H3K9和H3K27结合, 同时chromodomain也可识别非组蛋白和特定的核酸结构。不同类型的chromodomain蛋白在基因转录调节、基因组重排修复和染色质重塑等过程中发挥重要调控作用, 从多个层次参与染色质表观遗传调节过程。本文综述chromodomain的分类和结构特征, 探讨进化中不同的chromodomain蛋白在细胞中的功能多样性, 为进一步研究chromodomain蛋白在细胞中的作用机制提供参考。     

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase

Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The elongated structure of the conserved core of yeast Dot1p contains an N-terminal helical domain and a seven-stranded catalytic domain that harbors the binding site for the methyl-donor and an active site pocket sided with conserved hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended conformation distinct from the folded conformation observed in structures of SET domain histone lysine methyltransferases. A catalytic asparagine (Asn479), located at the bottom of the active site pocket, suggests a mechanism similar to that employed for amino methylation in DNA and protein glutamine methylation. The acidic, concave cleft between the two domains contains two basic residue binding pockets that could accommodate the outwardly protruding basic side chains around Lys79 of histone H3 on the disk-like nucleosome surface. Biochemical studies suggest that recombinant Dot1 proteins are active on recombinant nucleosomes, free of any modifications.     

Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix

The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.     

Superdomains in the protein structure hierarchy: The case of PTP-C2

Superdomain is uniquely defined in this work as a conserved combination of different globular domains in different proteins. The amino acid sequences of 25 structurally and functionally diverse proteins from fungi, plants, and animals have been analyzed in a test of the superdomain hypothesis. Each of the proteins contains a protein tyrosine phosphatase (PTP) domain followed by a C2 domain. Four novel conserved sequence motifs have been identified, one in the PTP domain and three in the C2 domain. All contribute to the PTP-C2 domain interface in PTEN, a tumor suppressor, and all are more conserved than the PTP signature motif, HCX₃(K/R)XR, in the 25 sequences. We show that PTP-C2 was formed prior to the fungi, plant, and animal kingdom divergence. A superdomain as defined here does not fit the usual protein structure classification system. The demonstrated existence of one superdomain suggests the existence of others.     

Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function

The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.     

The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins,APETALA3 and PISTILLATA

MADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.     

NrdH-redoxin of Corynebacterium ammoniagenes forms a domain-swapped dimer

NrdH-redoxins constitute a family of small redox proteins, which contain a conserved CXXC sequence motif, and are characterized by a glutaredoxin-like amino acid sequence but a thioredoxin-like activity profile. Here we report the structure of Corynebacterium ammoniagenes NrdH at 2.7 A resolution, determined by molecular replacement using E. coli NrdH as model. The structure is the first example of a domain-swapped dimer from the thioredoxin family. The domain-swapped structure is formed by an inter-chain two-stranded anti-parallel beta-sheet and is stabilized by electrostatic interactions at the dimer interface. Size exclusion chromatography, and MALDI-ESI experiments revealed however, that the protein exists as a monomer in solution. Similar to E. coli NrdH-redoxin and thioredoxin, C. ammoniagenes NrdH-redoxin has a wide hydrophobic pocket at the surface that could be involved in binding to thioredoxin reductase. However, the loop between alpha2 and beta3, which is complementary to a crevice in the reductase in the thioredoxin-thioredoxin reductase complex, is the hinge for formation of the swapped dimer in C. ammoniagenes NrdH-redoxin. C. ammoniagenes NrdH-redoxin has the highly conserved sequence motif W61-S-G-F-R-P-[DE]67 which is unique to the NrdH-redoxins and which determines the orientation of helix alpha3. An extended hydrogen-bond network, similar to that in E. coli NrdH-redoxin, determines the conformation of the loop formed by the conserved motif.     

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.     

Amino acid substitutions in a long flexible sequence influence thermodynamics and internal dynamic properties of winged helix protein genesis and its DNA complex

The hepatocyte nuclear factor (HNF)-3 homologous DNA binding domain is a highly conserved motif that contains a well-folded helix-turn-helix motif and two highly dynamic wings. Although the function and the properties of this motif have been intensively studied, the role of the internal wing (wing 1) is not well understood. In this study, amino acid substitutions were introduced into wing 1 of a conserved HNF-3 homologous protein, Genesis, and heteronuclear NMR, circular dichroism, DNA gel-shift assay, and fluorescent methods were employed to study and compare the properties of both wild-type and variant Genesis proteins. The data indicate that even though the substitutions are located on a dynamic wing outside the hydrophobic core sequences, they still globally influence biophysical properties of DNA-free Genesis and its DNA complex.     

Binding of the globular domain of linker histones H5/H1 to the nucleosome: a hypothesis

The amino acid sequence of the central globular domain of histone H1/H5 family members is highly homologous. Twenty-four such sequences have been compared to establish the conserved and variable residues. Fitting this to the tertiary structure of the H5 globular domain shows which of the conserved and variable residues are peripheral and which internal. Particular attention is paid to conserved basic residues on the surface, which we take to be DNA binding. Variable regions and conserved acidic residues are assumed not to be sites of contact with DNA. We conclude that one face of the domain, containing a cluster of basic residues, is the principal DNA binding site whilst two opposing faces, orthogonal to the principal site and also containing conserved basic residues, are subsidiary DNA binding sites. Since the DNA binding surface of the domain covers a full 180 degrees arc, we propose that it contacts a 'cage' of three DNA strands on the 2-fold axis of the chromatosome.