Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-ΔC) was solved in an oligomeric state. The molecular structure of BMRF1-ΔC shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-ΔC, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-ΔC exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-ΔC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-ΔC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-ΔC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.     

Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer.

Human sex hormone-binding globulin (SHBG) transports sex steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its steroid binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single steroid-binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and steroid-binding sites are distinct and that both monomers within a homodimeric complex are capable of binding steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a steroid-binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical steroid binding capacities. These data indicate that both subunits of the SHBG homodimer bind steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.     

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.     

IL-8 single-chain homodimers and heterodimers: interactions with chemokine receptors CXCR1, CXCR2, and DARC.

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants.     

Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis

The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Ų. TIMs from Trichomonas vaginalis (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.     

The DNA binding domain of a papillomavirus E2 protein programs a chimeric nuclease to cleave integrated human papillomavirus DNA in HeLa cervical carcinoma cells

Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.     

Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties

We have investigated the influence of the N-terminal domain of the 94-kDa glucocorticoid receptor on the DNA:receptor interaction. An alpha-chymotrypsin-induced 39-kDa receptor fragment, containing the hormone and DNA binding domains, binds DNA with a reduced specificity compared to the intact 94-kDa receptor. Various footprinting assays did not reveal any qualitative differences when comparing the DNA contact points made by the two different receptor entities. Like the intact receptor, the 39-kDa receptor fragment binds as a dimer to DNA. Glutaraldehyde cross-linking demonstrated a difference in the protein:protein contacts of the two homodimers. Furthermore, the dimeric 94-kDa receptor did not recognize a half-DNA site, while the dissociated 94-kDa receptor dimer and the dimeric 39-kDa receptor fragment allowed binding to such a site. These results suggest that the loss of the N-terminal domain of the receptor affects the steric arrangement and/or rigidity of the two DNA binding domains of the receptor homodimer, resulting in a decreased DNA binding specificity of the 39-kDa receptor fragment.     

Dimer formation by a "monomeric" protein

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 10(5)-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 degrees C results in a 10(3)-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 degrees C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer-dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 degrees C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of "monomeric" proteins could be more prevalent than is usually appreciated.     

Single chain dimers of MASH-1 bind DNA with enhanced affinity.

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.     

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.     

The papillomavirus E8-E2C protein represses DNA replication from extrachromosomal origins

Carcinogenic DNA viruses such as high-risk human papillomaviruses (HPV) and Epstein-Barr-Virus (EBV) replicate during persistent infections as low-copy-number plasmids. EBV DNA replication is restricted by host cell replication licensing mechanisms. In contrast, copy number control of HPV genomes is not under cellular control but involves the viral sequence-specific DNA-binding E2 activator and E8-E2C repressor proteins. Analysis of HPV31 mutant genomes revealed that residues outside of the DNA-binding/dimerization domain of E8-E2C limit viral DNA replication, indicating that binding site competition or heterodimerization among E2 and E8-E2C proteins does not contribute to copy number control. Domain swap experiments demonstrated that the amino-terminal 21 amino acids of E8-E2C represent a novel, transferable DNA replication repressor domain, whose activity requires conserved lysine and tryptophan residues. Furthermore, E8-E2C (1-21)-GAL4 fusion proteins inhibited the replication of the plasmid origin of replication of EBV, suggesting that E8-E2C functions as a general replication repressor of extrachromosomal origins. This finding could be important for the development of novel therapies against persistent DNA tumor virus infections.     

DNA sequence recognition by bispyrazinonaphthalimides antitumor agents

Bifunctional DNA intercalating agents have long attracted considerable attention as anticancer agents. One of the lead compounds in this category is the dimeric antitumor drug elinafide, composed of two tricyclic naphthalimide chromophores separated by an aminoalkyl linker chain optimally designed to permit bisintercalation of the drug into DNA. In an effort to optimize the DNA recognition capacity, different series of elinafide analogues have been prepared by extending the surface of the planar drug chromophore which is important for DNA sequence recognition. We report here a detailed investigation of the DNA sequence preference of three tetracyclic monomeric or dimeric pyrazinonaphthalimide derivatives. Melting temperature measurements and surface plasmon resonance (SPR) studies indicate that the dimerization of the tetracyclic planar chromophore considerably augments the affinity of the drug for DNA, polynucleotides, or hairpin oligonucleotides and promotes selective interaction with G.C sites. The (CH(2))(2)NH(CH(2))(3)NH(CH(2))(2) connector stabilizes the drug-DNA complexes. The methylation of the two nitrogen atoms of this linker chain reduces the binding affinity and increases the dissociation rates of the drug-DNA complexes by a factor of 10. DNase I footprinting experiments were used to investigate the sequence selectivity of the drugs, demonstrating highly preferential binding to G.C-rich sequences. It also served to select a high-affinity site encompassing the sequence 5'-GACGGCCAG which was then introduced into a biotin-labeled hairpin oligonucleotide to accurately measure the binding parameters by SPR. The affinity constant of the unmethylated dimer for this sequence is 500 times higher than that of the monomer compound and approximately 10 times higher than that of the methylated dimer. The DNA groove accessibility was also probed with three related oligonucleotides carrying G --> c(7)G, G --> I, and C --> M substitutions. The level of drug binding to the two hairpin oligonucleotides containing 7-deazaguanine (c(7)G) or 5-methylcytosine (M) residues is unchanged or only slightly reduced compared to that of the unmodified target. In contrast, incorporation of inosine (I) residues considerably decreases the extent of drug binding or even abolishes the interaction as is the case with the monomer. The pyrazinonaphthalimide derivatives are thus much more sensitive to the deletion of the exocyclic guanine 2-amino group exposed in the minor groove of the duplex than to the modification of the major groove elements. The complementary SPR footprinting methodology combining site selection and quantitative DNA affinity analysis constitutes a reliable method for dissecting the DNA sequence selectivity profile of reversible DNA binding small molecules.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.