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PubMed1996

Inhibition of transcription factor binding by ultraviolet-induced pyrimidine dimers.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for the induction of mutations and the development of skin cancer. Cis-syn cyclobutane pyrimidine dimers (pyrimidine dimers) are the most frequent lesions produced in DNA by UV irradiation. Besides being mutagenic, pyrimidine dimers may interfere with other important DNA-dependent processes. To analyze the effects of pyrimidine dimers on the ability of DNA sequences to be recognized by trans-acting factors, we have incorporated site-specific T-T dimers into oligonucleotides containing the recognition sequences of the sequence-specific transcription factors E2F, NF-Y, AP-1, NF kappa B, and p53. In each case, presence of the photodimer strongly inhibited binding of the respective transcription factor complex. Reduction of binding varied between 11- and 60-fold. The results indicate that the most common UV-induced DNA lesion can interfere severely with binding of several important cell cycle regulatory and DNA damage responsive transcription factors. We suggest that inhibition of transcription factor binding may be a major biological effect of UV radiation since promoter regions are known to be repaired inefficiently and since UV damage can deregulate the function of a large number of different factors.

Binding Sites↗
PubMed1998

Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1.

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

Binding Sites↗
PubMed1991

Pyrimidine dimer induction and repair in cultured human skin keratinocytes or melanocytes after irradiation with monochromatic ultraviolet radiation.

We compared the susceptibilities of cultured melanocytes and keratinocytes to dimer induction in DNA by monochromatic ultraviolet (UV) radiation. Keratinocytes as well as melanocytes were derived from human foreskin, grown as a monolayer in petri dishes, covered with phosphate-buffered saline containing 0.1% glucose, and irradiated. UV irradiation was carried out at 254, 297, and 302 nm as well as with a light source emitting predominantly 312 nm. The induction of pyrmidine dimers was assessed by determination of the number of T4 endonuclease V-sensitive sites (ESS). We found a slightly higher response for dimer induction in melanocytes at 254, 297, and 302 nm; this difference was only significant at the 297-nm wavelength. Action spectra for pyrimidine dimer induction were derived from the exposure-response data obtained. The action spectra mimic to a large degree the action spectra for dimer induction in other cultured mammalian cells. The repair rate during a post-irradiation period lasting up to 24 h was substantially the same for the two cell types. The percentage of T4 endonuclease V-sensitive sites (ESS) remaining 9 and 24 h after irradiation was 45% and 30%, respectively.

Cells, Cultured↗
PubMed1998

Role of quaternary structure in muscle creatine kinase stability: tryptophan 210 is important for dimer cohesion.

A mutant of the dimeric rabbit muscle creatine kinase (MM-CK) in which tryptophan 210 was replaced has been studied to assess the role of this residue in dimer cohesion and the importance of the dimeric state for the native enzyme stability. Wild-type protein equilibrium unfolding induced by guanidine hydrochloride occurs through intermediate states with formation of a molten globule and a premolten globule. Unlike the wild-type enzyme, the mutant inactivates at lower denaturant concentration and the loss of enzymatic activity is accompanied by the dissociation of the dimer into two apparently compact monomers. However, the Stokes radius of the monomer increases with denaturant concentration as determined by size exclusion chromatography, indicating that, upon monomerization, the protein structure is destabilized. Binding of 8-anilinonaphthalene-1-sulfonate shows that the dissociated monomer exposes hydrophobic patches at its surface, suggesting that it could be a molten globule. At higher denaturant concentrations, both wild-type and mutant follow similar denaturation pathways with formation of a premolten globule around 1.5-M guanidine, indicating that tryptophan 210 does not contribute to a large extent to the monomer conformational stability, which may be ensured in the dimeric state through quaternary interactions.

Anilino Naphthalenesulfonates↗
PubMed1997

Functional interaction between Chlamydomonas outer arm dynein subunits: the gamma subunit suppresses the ATPase activity of the alpha beta dimer.

The alpha beta dimer and the gamma subunit of the Chlamydomonas outer arm dynein were solubilized by treating isolated axonemes with 0.6 M KCI, and purified by sucrose density gradient centrifugation. The axonemes were from an ida1 mutant to eliminate contamination of outer arm subunits by inner arm dynein 11, and the axonemes were pre-extracted with 0.6 M CH3COOK to remove non-dynein protein that might otherwise contaminate outer arm dynein fractions in the sucrose gradient. In addition, purer fractions of outer arm dynein subunits were obtained by modifying the centrifugation conditions to take advantage of the propensity of the dynein to dissociate under high hydrostatic pressure in the presence of Mg2+. When sucrose gradient fractions containing the gamma subunit were added to a fraction containing the purified alpha beta dimer under conditions expected to promote reassociation of the subunits to form a trimeric outer arm dynein complex [Takada et al., 1992: J. Biochem, 111:758-762], the total ATPase activity of the mixture was suppressed to a level lower than that of the original alpha beta dimer fraction. The inhibition paralleled the distribution of gamma subunit in the sucrose gradient, was saturable, and was maximum at an approximately equimolar ratio of the gamma subunit to the alpha beta dimer. These results indicate that when the gamma subunit interacts with the alpha beta dimer, the latter's ATPase activity is modulated downward. Previous results showed that interaction of the alpha subunit with the beta subunit suppressed the beta subunit's ATPase activity [Pfister and Witman, 1984: J. Biol. Chem. 259:12072-12080]. Thus, the total ATPase activity of the outer arm dynein is dependent upon communication between all three subunits within the arm.

Adenosine Triphosphatases↗
PubMed1997

Molecular modeling of c-erbB2 receptor dimerization: coiled-coil structure of wild and oncogenic transmembrane domains--stabilization by interhelical hydrogen bonds in the oncogenic form.

Dimerization models of c-erbB2 transmembrane domains (Leu651-Ile675) are studied by molecular mechanics and molecular dynamics simulations. Both wild and Glu mutated transmembrane helices exhibit the same relative orientation for favorable associations and dimerize preferentially in left-handed coiled-coil structures. The mutation point 659 belongs to the interfacing residues, and in the transforming domain, symmetric hydrogen bonds between Glu carboxylic groups stabilize the dimeric structure. The same helix packing found for the wild dimers, except side-chain-side-chain hydrogen bonds, suggests that the transmembrane domains dimerize according to similar process. Structural and energetical characterization of the models are presented.

Amino Acid Sequence↗
PubMed1999

Domain deletions in the human polymeric Ig receptor disclose differences between its dimeric IgA and pentameric IgM interaction.

The human polymeric Ig receptor (pIgR), or transmembrane secretory component, is basolaterally expressed on secretory epithelial cells; its function is to transport externally J chain-containing dimeric IgA and pentameric IgM. The ligand-binding extracellular part of this receptor contains five disulfide-stabilized domains which show considerable homology with the variable domains of Ig chains. The N-terminal domain 1 (D1) mediates the initial noncovalent ligand interaction. In this study we made deletions of the human pIgR D2 and D3 (pIgRDelta2,3), or D4 and D5 (pIgRDelta4,5), to investigate the influence of these domains in receptor binding and transport of dimeric IgA and pentameric IgM across MDCK cells transfected with the truncated receptors. Both mutants were found to bind pentameric IgM, but only pIgRDelta4,5 bound dimeric IgA. These results showed that the two ligands interact differently with human pIgR; binding of pentameric IgM apparently depends fully on strong interactions with D1, while binding of dimeric IgA in addition depends on elements within D2 and / or D3 to support the initial noncovalent binding to D1. Moreover, our studies imply that dimeric human IgA binds differently to pIgR from various species. This observation cautions against interpretation of functional studies performed with non-homologous receptor-ligand pairs.

Animals↗
PubMed2007

Effects of dimerization of Serratia marcescens endonuclease on water dynamics.

The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.

Binding Sites↗
PubMed2005

Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration.

This article examines the feasibility of using ultrafiltration to separate the monomer of the monoclonal antibody alemtuzumab (Campath or Campath-1H) from a mixture of dimer and higher-order oligomers (collectively called "dimers" here). Using parameter scanning ultrafiltration, we initially assessed the suitability of the following membranes: 100 kDa and 300 kDa polyethersulfone (PES) membranes, and a 100 kDa polyvinylidene fluoride (PVDF) membrane. A detailed study was then carried out to examine the effects of operating conditions (such as solution pH, ionic strength, stirring speed, and permeate flux) on the separation of the monomer from the dimers using 300 kDa PES and 100 kDa PVDF membranes. Results of the experiments carried out in the carrier phase ultrafiltration (CPUF) mode indicate that the size-based protein-protein separation critically depends on the membrane used as well as the system hydrodynamics. The separation of the monoclonal antibody monomer and dimers using 100 kDa PVDF membranes in the diafiltration mode was also examined. Experimental results demonstrate that under suitable conditions, it is feasible to obtain the alemtuzumab monomer with a purity of more than 93% and a yield of more than 85% (from a mixture of 75% monomer and 25% dimers, which is the typical composition obtained after affinity chromatography). Simulation study indicates that this could be further improved to a purity of more than 96% and a monomer yield of more than 96% by increasing the selectivity of separation or by employing a two-stage diafiltration process.

Alemtuzumab↗
PubMed2004

Amphotericin B covalent dimers bearing a tartarate linkage.

Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 microM) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB.

Amphotericin B↗
PubMed2006

Twist1 dimer selection regulates cranial suture patterning and fusion.

Saethre-Chotzen syndrome is associated with haploinsufficiency of the basic-helix-loop-helix (bHLH) transcription factor TWIST1 and is characterized by premature closure of the cranial sutures, termed craniosynostosis; however, the mechanisms underlying this defect are unclear. Twist1 has been shown to play both positive and negative roles in mesenchymal specification and differentiation, and here we show that the activity of Twist1 is dependent on its dimer partner. Twist1 forms both homodimers (T/T) and heterodimers with E2A E proteins (T/E) and the relative level of Twist1 to the HLH inhibitor Id proteins determines which dimer forms. On the basis of the expression patterns of Twist1 and Id1 within the cranial sutures, we hypothesized that Twist1 forms homodimers in the osteogenic fronts and T/E heterodimers in the mid-sutures. In support of this hypothesis, we have found that genes regulated by T/T homodimers, such as FGFR2 and periostin, are expressed in the osteogenic fronts, whereas genes regulated by T/E heterodimers, such as thrombospondin-1, are expressed in the mid-sutures. The ratio between these dimers is altered in the sutures of Twist1+/- mice, favoring an increase in homodimers and an expansion of the osteogenic fronts. Of interest, the T/T to T/E ratio is greater in the coronal versus the sagittal suture, and this finding may contribute to making the coronal suture more susceptible to fusion due to TWIST haploinsufficiency. Importantly, we were able to inhibit suture fusion in Twist1+/- mice by modulating the balance between these dimers toward T/E formation, by either increasing the expression of E2A E12 or by decreasing Id expression. Therefore, we have identified dimer partner selection as an important mediator of Twist1 function and provide a mechanistic understanding of craniosynostosis due to TWIST haploinsufficiency.

Animals↗
PubMedDate not supplied

Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems.

BACKGROUND: Exogenously controlled gene expression systems are essential for both the in vivo analysis of gene function and the regulated delivery of therapeutic gene products. However, differences in experimental methods used to characterize the various systems prohibit informative comparisons. The purpose of this study was to identify an optimal system for regulated gene expression studies through a rigorous direct comparison of the dimerizer and the reverse tet transactivator (rtTA) transcriptional switch systems. METHODS: An optimized bicistronic rapamycin-dependent dimerizer construct and an optimized rtTA construct (based on rtTA(s)-M2) were developed that utilize a chimeric mammalian activation domain with a flexible interdomain linker. These constructs were reconstituted in identical eukaryotic expression vectors and compared in transient transfection assays employing target gene reporter constructs that differ only in the relevant DNA binding sites. RESULTS: The optimized rtTA(s)-M2 construct, designated rtTAM2.2, exhibited a twofold increase in the magnitude of doxycycline-dependent reporter gene induction and an eightfold increase in sensitivity as compared to the rtTA(s)-M2 construct. This correlated with a significantly higher level of expression of the rtTAM2.2 protein. In direct comparisons the rtTAM2.2 system mediated inducible expression to a level tenfold greater than the bicistronic dimerizer system. However, while the dimerizer system exhibited no detectable rapamycin-independent expression, a low level of doxycycline-independent target gene expression was detectable. CONCLUSIONS: The improved rtTAM2.2 rtTA system described here may prove optimal when the overall magnitude of target gene induction is critical, while the dimerizer system may be advantageous when the complete absence of ligand-independent target gene expression is essential.

Animals↗
PubMed2005

Structure determination and conformation analysis of symmetrical dimers.

Conformational and stereochemical analysis of six new symmetrical dimers was performed using proton-proton vicinal coupling measured from (1)H NMR and (13)C satellites of (1)H NMR signals, natural abundance (13)C-edited nuclear overhauser effect (NOE) experiments, comprehensive NOE analysis and molecular modeling. The (13)C satellite analysis and (13)C-edited NOE experiments were carried out to extract spectral information between equivalent protons. Molecular modeling was applied for estimations of three-dimensional parameters of the studied dimers, which were subsequently used to generate a set of theoretical NOE for each possible conformation. The J-coupling, (13)C-edited NOE and quantitative NOE analyses showed the predominance of gauche conformation for three dimers, whereas a mixture of gauche and anti conformations (45:55) for three other dimers was established by quantitative NOE analysis. X-ray crystallographic study confirmed the stereochemistry of one of the dimers and revealed a discrepancy in conformation stability between liquid and solid states.

Carbon Isotopes↗
PubMed2002

Design and folding of dimeric proteins.

In a similar way in which the folding of single-domain proteins provides an important test in the study of self-organization, the folding of homodimers constitutes a basic challenge in the quest for the mechanisms that are the basis of biological recognition. Dimerization is studied by following the evolution of two identical 20-letter amino acid chains within the framework of a lattice model and using Monte Carlo simulations. It is found that when design (evolution pressure) selects few, strongly interacting (conserved) amino acids to control the process, a three-state folding scenario follows, where the monomers first fold forming the halves of the eventual dimeric interface independently of each other, and then dimerize ("lock and key" kind of association). On the other hand, if design distributes the control of the folding process on a large number of (conserved) amino acids, a two-state folding scenario ensues, where dimerization takes place at the beginning of the process, resulting in an "induced type" of association. Making use of conservation patterns of families of analogous dimers, it is possible to compare the model predictions with the behavior of real proteins. It is found that theory provides an overall account of the experimental findings.

Amino Acid Sequence↗
PubMed2001

Computation and mutagenesis suggest a right-handed structure for the synaptobrevin transmembrane dimer.

Biological membrane fusion involves a highly precise and ordered set of protein-protein interactions. Synaptobrevin is a key player in this process. Mutagenesis studies of its single transmembrane segment suggest that it dimerizes in a sequence specific manner. Using the computational methods developed for the successful structure prediction of the glycophorin A transmembrane dimer, we have calculated a structural model for the synaptobrevin dimer. Our computational search yields a well-populated cluster of right-handed structures consistent with the experimentally determined dimerization motif. The three-dimensional structure contains an interface formed primarily by leucine and isoleucine side-chain atoms and has no interhelical hydrogen bonds. The model is the first three-dimensional picture of the synaptobrevin transmembrane dimer and provides a basis for further focused experimentation on its structure and association thermodynamics.

Algorithms↗
PubMed2005

Crystal structure of a calcium-induced dimer of two isoforms of cobra phospholipase A2 at 1.6 A resolution.

The calcium-induced formation of a complex between two isoforms of cobra venom phospholipase A2 reveals a novel interplay between the monomer-dimer and activity-inactivity transitions. The monodispersed isoforms lack activity in the absence of calcium ions while both molecules gain activity in the presence of calcium ions. At concentrations higher than 10 mg/ml, in the presence of calcium ions, they dimerize and lose activity again. The present study reports the crystal structure of a calcium-induced dimer between two isoforms of cobra phospholipase A2. In the complex, one molecule contains a calcium ion in the calcium binding loop while the second molecule does not possess an intramolecular calcium ion. However, there are two calcium ions per dimer in the structure. The second calcium ion is present at an intermolecular site and that is presumably responsible for the dimerization. The calcium binding loops of the two molecules adopt strikingly different conformations. The so-called calcium binding loop in the calcium-containing molecule adopts a normal conformation as generally observed in other calcium containing phospholipase A(2) enzymes while the conformation of the corresponding loop in the calcium free monomer deviates considerably with the formation of a unique intraloop Gly33 (N)-Cys27 (O) = 2.74 A backbone hydrogen bond. The interactions of Arg31 (B) with Asp49 (A) and absence of calcium ion are responsible for the loss of catalytic activity in molecule A while interactions of Arg2 (B) with Tyr52 (B) inactivate molecule B.

Animals↗
PubMed2000

Structural requirements for catalysis and dimerization of human methionine adenosyltransferase I/III.

We have used site-directed mutagenesis to probe the structural requirements for catalysis and dimerization of human hepatic methionine adenosyltransferase (hMAT). We built a homology model of the dimeric hMAT III inferred by the crystal structure of the highly homologous Escherichia coli MAT dimer. The active sites of both enzymes comprise the same amino acids and are located in the inter-subunit interface. All of the amino acids predicted to be in the hMAT III active site were mutated, as well as residues in a conserved ATP binding region. All of the mutations except one severely affected catalytic activity. On the other hand, dimerization was affected only by single mutations of three different residues, all on one monomer. The homology model suggested that the side chains of these residues stabilized the monomer and participated in a bridge between subunits consisting of a network of metal and phosphate ions. In agreement with this observation, we demonstrated that dimerization cannot occur in the absence of phosphate.

Amino Acid Sequence↗
PubMed1998

FGF-2 dimerization involvement in growth factor mediated cell proliferation but not cell differentiation.

Dimerization is a prerequisite for many growth factors in their receptor activation leading to cellular response. FGF-1 and FGF-2, members of the Fibroblast Growth Factor (FGF) family, were shown to form non-covalent dimers and oligomers in vitro. Using the two-hybrid system as an in vivo binding assay we show here that of three representative members of the FGF family, only FGF-2 is able to homodimerize. Moreover the FGF-2 isoforms could heterodimerize. Two single-point mutants (T121F and W123R), defective in their dimerization capability, were isolated through random mutagenesis and were used to study the role of FGF-2 dimerization with regard to its biological activity. Remarkably, these mutant proteins were still able to induce cell differentiation, but were strongly affected in their capacity to promote cell proliferation. This study thus highlights the uncoupling between proliferation and differentiation FGF-2 signaling pathways and the crucial role of FGF-2 dimerization in the mitogenic activity of this factor.

Animals↗