Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.     

Genetic analysis of B55alpha/Cdc55 protein phosphatase 2A subunits: association with the adenovirus E4orf4 protein

The human adenovirus E4orf4 protein is toxic in both human tumor cells and Saccharomyces cerevisiae. Previous studies indicated that most of this toxicity is dependent on an interaction of E4orf4 protein with the B55 class of regulatory subunits of protein phosphatase 2A (PP2A) and in yeast with the B55 homolog Cdc55. We have found previously that E4orf4 inhibits PP2A activity against at least some substrates. In an attempt to understand the mechanism of this inhibition, we used a genetic approach to identify residues in the seven-bladed β-propeller proteins B55α and Cdc55 required for E4orf4 binding. In both cases, amino-terminal polypeptides composed only of blade 1 and at least part of blade 2 were found to bind E4orf4 and overexpression blocked E4orf4 toxicity in yeast. Furthermore, certain amino acid substitutions in blades 1 and 2 within full-length B55α and Cdc55 resulted in loss of E4orf4 binding. Recent mutational analysis has suggested that segments of blades 1 and 2 present on the top face of B55α form part of the "substrate-binding groove." Additionally, these segments are in close proximity to the catalytic C subunit of the PP2A holoenzyme. Thus, our results are consistent with the hypothesis that E4orf4 binding could affect the access of substrates, resulting in the failure to dephosphorylate some PP2A substrates.     

Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line

The human adenovirus E4 ORF 6 34 kDa oncoprotein (E4 34k), in concert with the 55 kDa product of E1b, prevents concatenation of viral genomes in infected cells, inhibits the repair of double strand breaks (DSBs) in the viral genome, and inhibits V(D)J recombination in a plasmid transfection assay. These activities are consistent with a general inhibition by the E4 34k and E1b 55k proteins of DSB repair by non-homologous end joining (NHEJ) on extrachromosomal substrates. To determine whether inhibition of NHEJ extends to repair of DSBs in the cell chromosome, we have examined the effects of E4 34k on repair of chromosomal DSBs induced by ionizing radiation in a cell line in which E4 34k expression and biological activity is inducible and E1b 55k is produced constitutively. We demonstrate that in this cell line, induction of E4 34k inhibits chromosomal DSB repair. Recently, it has been shown that in infected cells, E4 34k and the adenovirus E1b 55k proteins cooperate to destabilize Mre11 and Rad50, components of mammalian NHEJ systems. Consistent with this, induction of expression of E4 34k in the inducible cell line also reduces the steady state level of Mre11 protein.     

Induction of apoptosis by adenovirus E4orf4 protein

Adenovirus E4orf4 protein is a multifunctional viral regulator that induces p53-independent apoptosis in transformed cells, but not in normal cells. E4orf4-induced apoptosis can occur without activation of known caspases, although E4orf4 induces caspase activity in some cell lines. The interaction of E4orf4 with a specific subpopulation of protein phosphatase 2A (PP2A) molecules that contain B subunits, but not with those that contain B subunits, is required for induction of apoptosis. This review suggests the potential use of E4orf4 in cancer therapy, and discusses whether E4orf4-induced apoptosis plays a role in the viral life cycle. Future research directions are also highlighted.     

Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.     

Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orf6 protein

Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino- and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxy-terminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal "oncodomain" of the viral protein.     

A zinc-binding protein in a metal-resistant strain, Alcaligenes eutrophus CH34.

Synthesis of a zinc-binding protein was induced when Alcaligenes eutrophus CH34 was grown in the presence of high concentrations of zinc (2.3 mM). The zinc-binding protein has a low content of cysteine and a high content of acidic amino acids and, thus, differs from metallothionein.     

Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.     

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex.

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa EIB protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.     

The adenovirus E4-ORF4 splicing enhancer protein interacts with a subset of phosphorylated SR proteins

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.     

The protein conformation and a zinc-binding domain of an autoantigen from mouse seminal vesicle.

The protein conformation of a mouse seminal vesicle autoantigen was studied by circular dichroism spectroscopy. At pH 7.4, the spectrum in the UV region appears as one negative band at 217 nm and one positive band at 200 nm. This together with the predicted secondary structures indicates no helices but a mixture of beta form, beta turn, and unordered form in the protein molecule. The conformation is stable even at pH 10.5 or 3.0. The spectrum in the near-UV region consists of fine structures that are disturbed in acidic or alkaline solution. The environments around Trp2 and Trp82 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 345 nm, and about 87% of them are accessible to quenching by acrylamide. Correlating the quenching effect of CsCl and Kl on the protein fluorescence to the charged groups along the polypeptide chain suggests the difference in the "local charge" around the two tryptophan residues. The presence of ZnCl2 in the protein solution effects no change in the circular dichroism but perturbs the fluorescence due to Trp82. Analysis of the fluorescence data suggests a Zn(2+)-binding site on the protein, which cannot coordinate with both Ca2+ and Mg2+. The association constant for the complex formation is 1.35 x 10(5) +/- 0.04 x 10(5) M-1 at pH 7.4.     

Novel topology of a zinc-binding domain from a protein involved in regulating early Xenopus development.

Xenopus nuclear factor XNF7, a maternally expressed protein, functions in patterning of the embryo. XNF7 contains a number of defined protein domains implicated in the regulation of some developmental processes. Among these is a tripartite motif comprising a zinc-binding RING finger and B-box domain next to a predicted alpha-helical coiled-coil domain. Interestingly, this motif is found in a variety of protein including several proto-oncoproteins. Here we describe the solution structure of the XNF7 B-box zinc-binding domain determined at physiological pH by 1H NMR methods. The B-box structure represents the first three-dimensional structure of this new motif and comprises a monomer have two beta-strands, two helical turns and three extended loop regions packed in a novel topology. The r.m.s. deviation for the best 18 structures is 1.15 A for backbone atoms and 1.94 A for all atoms. Structure calculations and biochemical data shows one zinc atom ligated in a Cys2-His2 tetrahedral arrangement. We have used mutant peptides to determine the metal ligation scheme which surprisingly shows that not all of the seven conserved cysteines/histidines in the B-box motif are involved in metal ligation. The B-box structure is not similar in tertiary fold to any other known zinc-binding motif.     

Expression of a zinc-binding domain of boar spermatidal transition protein 2 in Escherichia coli.

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc.     

Structure of a zinc-binding domain in the Junin virus envelope glycoprotein

Arenaviruses cause acute hemorrhagic fevers with high mortality. Entry of the virus into the host cell is mediated by the viral envelope glycoprotein, GPC. In contrast to other class I viral envelope glycoproteins, the mature GPC complex contains a cleaved stable signal peptide (SSP) in addition to the canonical receptor-binding (G1) and transmembrane fusion (G2) subunits. SSP is critical for intracellular transport of the GPC complex to the cell surface and for its membrane-fusion activity. Previous studies have suggested that SSP is retained in GPC through interaction with a zinc-binding domain (ZBD) in the cytoplasmic tail of G2. Here we used NMR spectroscopy to determine the structure of Junín virus (JUNV) ZBD (G2 residues 445-485) and investigate its interaction with a conserved Cys residue (Cys-57) in SSP. We show that JUNV ZBD displays a novel fold containing two zinc ions. One zinc ion is coordinated by His-447, His-449, Cys-455, and His-485. The second zinc ion is coordinated by His-459, Cys-467, and Cys-469 and readily accepts Cys-57 from SSP as the fourth ligand. Our studies describe the structural basis for retention of the unique SSP subunit and suggest a mechanism whereby SSP is positioned in the GPC complex to modulate pH-dependent membrane fusion.     

Specific recognition of four-way DNA junctions by the C-terminal zinc-binding domain of HPV oncoprotein E6

E6 is an oncoprotein implicated in cervical cancers produced by " high risk " human papillomaviruses. E6 binds specifically to several cellular proteins, including the tumour suppressor p53 and the ubiquitin ligase E6-AP. However, E6 is also a DNA-binding protein which recognizes a structural motive present in four-way junctions. Here, we demonstrate that the C-terminal zinc-binding domain of E6, expressed separately from the rest of the protein, fully retains the selective four-way junction recognition activity. The domain can bind to two identical and independent sites on a single junction, whereas full-length E6 can only bind to one site. The junction bound to either one or two domains adopts an extended square conformation. These results allow us to assign the structure-dependent DNA recognition activity of E6 to its C-terminal domain, which therefore represents a new class of zinc-stabilized DNA-binding module. Comparison with the binding characteristics of other junction-specific proteins enlightens the rules which govern protein-induced deformation of four-way DNA junctions.     

Structure of the adenovirus E4 Orf6 protein predicted by fold recognition and comparative protein modeling

To facilitate investigation of the molecular and biochemical functions of the adenovirus E4 Orf6 protein, we sought to derive three-dimensional structural information using computational methods, particularly threading and comparative protein modeling. The amino acid sequence of the protein was used for secondary structure and hidden Markov model (HMM) analyses, and for fold recognition by the ProCeryon program. Six alternative models were generated from the top-scoring folds identified by threading. These models were examined by 3D-1D analysis and evaluated in the light of available experimental evidence. The final model of the E4 protein derived from these and additional threading calculations was a chimera, with the tertiary structure of its C-terminal 226 residues derived from a TIM barrel template and a mainly alpha-nonbundle topology for its poorly conserved N-terminal 68 residues. To assess the accuracy of this model, additional threading calculations were performed with E4 Orf6 sequences altered as in previous experimental studies. The proposed structural model is consistent with the reported secondary structure of a functionally important C-terminal sequence and can account for the properties of proteins carrying alterations in functionally important sequences or of those that disrupt an unusual zinc-coordination motif.