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PubMed1997

Dimerization of the p185neu transmembrane domain is necessary but not sufficient for transformation.

The neu proto-oncogene encodes a receptor tyrosine kinase (RTK). The oncogenic allele neu* (p185*) bears a glutamic acid for valine substitution at position 664 within the predicted transmembrane domain. We have used this mutant to explore the role of the transmembrane domain in signal transduction by RTKs. Analysis of a panel of neu* proteins with second-site mutations in the transmembrane domain revealed a strong correlation of dimerization with transformation. Both dimerization and transformation are dependent on a domain formed by the amino acids Val663-Glu664-Gly665 (VEG). However, movement of the VEG elsewhere within the transmembrane domain promoted weak dimerization but not transformation. Epidermal growth factor receptor (EGFR)/neu chimeras were used to determine if mutations that disrupt activation by Glu664 affect hormone-regulated signal transduction as well. These mutations (of Val663 and Gly665) did not affect regulation by EGF. Introduction of the known transmembrane dimerization domain from Glycophorin A (GpA) stimulated dimerization, but was not sufficient for transformation. These results indicate that dimerization is necessary but not sufficient for transforming activity. The homologous wild-type domain, VVG, is not required for hormone-regulated signaling.

3T3 Cells↗
PubMed2001

Folding of beta pep-4 beta-sheet sandwich dimers and tetramers is influenced by aliphatic hydrophobic residues at the intersubunit interface.

For the designed peptide 33mer, beta pep-4, formation of beta-sheet structure [Ilyina, Roongta and Mayo (1997) Biochemistry 36, 5245--5250] is thermodynamically linked to self-association. Dimers and tetramers are stabilized by interactions between hydrophobic residues lying on the hydrophobic faces of the amphipathic monomer subunits. The present study investigates the effects on folding and self-association of the substitution of two key hydrophobic residues (Ile(20) and Val(22)) at the beta-sheet sandwich interface of beta pep-4. Single-site (I20L, I20V, I20A, V22L, V22I and V22A; where I20L corresponds to the substitution of Ile(20) with leucine etc.) and double-site (I20L/V22L and I20V/V22I) variants have been investigated. Like parent beta pep-4, all variants can form dimers and tetramers. NOESY data indicate that the overall beta-sheet fold and intersubunit beta-strand alignments are the same in all variant tetramers. CD data for all variants indicate mostly beta-sheet character in dimers and random coil character in monomers. Only for the V22I variant is the beta-sheet fold stabilized in the monomer state. Pulse-field gradient NMR-derived diffusion coefficients, measured as a function of peptide concentration, provide a means for deriving the distribution of monomer, dimer and tetramer states and, therefore, equilibrium association constants. Relative thermodynamic stabilities, which vary no more than approx. 0.5 kcal/mol (where 1 kcal identical with 4.184 kJ) from peptide to peptide, are I20V/V22I>I20V>I20L/V22L=beta pep-4 (Delta G(D) of 7.5 kcal/mol)=I20L=I20A>V22I>V22L>V22A for dimer formation and I20V>I20L/V22L>I20L>beta pep-4 (Delta G(T) of 6 kcal/mol)>V22I>I20V/V22I>V22L>I20A>V22A for tetramer formation. For the most part, dimer and/or tetramer stabilities are enhanced by the presence of valine and leucine and are attenuated by the presence of isoleucine and alanine.

Amino Acid Sequence↗
PubMed2004

Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells.

Signal transducer and activator of transcription 3 (Stat3) dimerization is commonly thought to be triggered by its tyrosine phosphorylation in response to interleukin-6 (IL-6) or other cytokines. Accumulating evidence from in vitro studies, however, suggests that cytoplasmic Stat3 may be associated with high-molecular-mass protein complexes and/or dimerize prior to its activation. To directly study Stat3 dimerization and subcellular localization upon cytokine stimulation, we used live-cell fluorescence spectroscopy and imaging microscopy combined with fluorescence resonance energy transfer (FRET). Stat3 fusion proteins with spectral variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were constructed and expressed in human hepatoma cells (HepG2) and human embryonic kidney cells (HEK-293). Like wild-type Stat3, the fusion proteins redistributed from a preferentially cytoplasmic to nuclear localization upon IL-6 stimulation and supported IL-6-dependent target gene expression. FRET studies in cells co-expressing Stat3-CFP and Stat3-YFP demonstrated that Stat3 dimers exist in the absence of tyrosine phosphorylation. IL-6 induced a 2-fold increase of this basal FRET signal, indicating that tyrosine phosphorylation either increases the dimer/monomer ratio of Stat3 or induces a conformational change of the dimer yielding a higher FRET efficiency. Studies using a mutated Stat3 with a non-functional src-homology 2 (SH2) domain showed that the SH2 domain is essential for dimer formation of phosphorylated as well as non-phosphorylated Stat3. Furthermore, our data show that visualization of normalized FRET signals allow insights into the spatiotemporal dynamics of Stat3 signal transduction.

Active Transport, Cell Nucleus↗
PubMed2005

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli.

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 microM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

ATP-Binding Cassette Transporters↗
PubMed1996

Evidence for regulated dimerization of cell-cell adhesion molecule (C-CAM) in epithelial cells.

C-CAM is a Ca(2+)-independent cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily. Addition of chemical cross-linkers to isolated rat liver plasma membranes, intact epithelial cells and purified preparations of C-CAM stabilized one major C-CAM-containing product whose apparent molecular mass was approximately twice that of the C-CAM monomer. The failure to detect additional proteins after cleavage of the cross-linked species demonstrated that C-CAM exists as non-covalently linked dimers both in solution and on the cell surface. Dimerization occurred to the same extent in adherent monolayers and in single cell populations, indicating that dimer formation was the result of cis-interactions within the membranes of individual cells. Using isoform-specific anti-peptide antibodies, both C-CAM1 and C-CAM2 were found to be involved in dimerization, forming predominantly homo-dimeric species. Both calmodulin and Ca2+ ionophore modulated the level of dimer formation, suggesting a role for regulated self-association in the functional activity of C-CAM.

Adenosine Triphosphatases↗
PubMed1998

Human MUC5AC mucin dimerizes in the rough endoplasmic reticulum, similarly to the MUC2 mucin.

Biosynthetic studies on the human MUC5AC mucin were performed by immunoprecipitations with antisera recognizing only the non-O-glycosylated apomucin in the colon adenocarcinoma cell line LS 174T. Pulse-chase studies and subcellular fractionations showed that MUC5AC formed dimers in the rough endoplasmic reticulum within 15 min of the initiation of biosynthesis. No non-O-glycosylated species larger than dimers were identified. The dimerization was N-glycosylation-dependent, because tunicamycin treatment significantly lowered the rate of dimerization. When the biosynthesis of MUC5AC apomucin was compared with that of MUC2 apomucin, also produced in the LS 174T cell line, both apomucins were assembled in similar ways with respect to their rates of dimerization with and without inhibition of N-glycosylation. No heterodimerization was observed between the human MUC5AC and the MUC2 apomucins despite the extensive sequence similarities in the positions of the cysteine residues in the C-termini proposed to be involved in mucin dimerization.

Amino Acid Sequence↗
PubMed2001

Coronin forms a stable dimer through its C-terminal coiled coil region: an implicated role in its localization to cell periphery.

BACKGROUND: Coronin is an actin-binding protein, which contains WD (Trp-Asp) repeats and a coiled-coil motif, and plays a role in regulating organization of the actin cytoskeletal network. Coronin localizes to the cell periphery, is involved in lamellipodium extension, and has an implicated role in cytokinesis, cell motility and phagocytosis. RESULTS: Our experiments with two different tagged forms of Xenopus coronin (Xcoronin) have shown that Xcoronin forms an oligomer. This oligomer complex is stable, resistant to 2.4 M NaCl, 0.6 M KI or 2 M urea. Physiochemical analysis of endogenous Xcoronin and the protein expressed in COS7 cells or in bacteria has revealed that the oligomer complex is an Xcoronin dimer. A C-terminal coiled-coil motif of Xcoronin is necessary and sufficient for the dimerization. Mutations in the coiled-coil motif generated dimerization deficient mutants of Xcoronin. Moreover, these mutant forms of Xcoronin failed to localize to the cell periphery, suggesting that dimerization is important for the proper subcellular localization of Xcoronin. CONCLUSION: Xcoronin forms a stable dimer via its C-terminal coiled-coil region. We propose that coronin dimerization is necessary for its proper subcellular localization and function.

Actins↗
PubMed2002

How fast are UV-dimers repaired in human skin DNA in situ?

Sheehan et al (2002) published a paper on UV-dimers in human skin and their repair with contradictions in their own data. In one experiment they applied two doses, 0.65 and 2 MED, but the apparent dimer levels, measured by a immunohistochemical technique, only increased in skin of type II (Fig 2a). The authors offered as explanation that the level of DNA damage would saturate. The dimer levels reached are at around 1 dimer per 1 million nucleotides, and there has been no evidence for saturation when a quantitative postlabeling technique has been used (Bykov et al, 1998). There is no reason for saturation until most of the thydmidyl-thymidine positions would be occupied in DNA, which, based on literature with other intrastrand cross-linkers, would require dimer levels 1 : 100 (Försti et al, 1989), or 10,000 times higher levels of dimer than that obtained by an irradiation at a MED dose.

DNA Repair↗
PubMed2005

Effects of frustration, confinement, and surface interactions on the dimerization of an off-lattice beta-barrel protein.

We study the effects of confinement, sequence frustration, and surface interactions on the thermodynamics of dimerization of an off-lattice minimalist beta-barrel protein using replica exchange molecular dynamics. We vary the degree of frustration of the protein by tuning the specificity of the hydrophobic interactions and investigate dimerization in confining spheres of different radii. We also investigate surface effects by tethering the first residue of one of the proteins to a uniformly repulsive surface. We find that increasing the confinement and frustration stabilize the dimer, while adding a repulsive surface decreases its stability. Different ensembles of structures, including properly dimerized and various partially dimerized states, are observed at the association transition temperature T(a), depending on the amount of frustration and whether a surface is present. The presence of a surface is predicted to alter the morphology of larger aggregates formed from partially unfolded dimeric conformations.

Chemistry, Physical↗
PubMed2004

Kinetic control of dimer structure formation in amyloid fibrillogenesis.

Amyloid fibril formation involves nonfibrillar oligomeric intermediates, which are important as possible cytotoxic species in neurodegenerative diseases. However, their transient nature and polydispersity have made it difficult to identify their formation mechanism or structure. We have investigated the dimerization process, the first step in aggregate formation, by multiple molecular dynamics simulations of five beta-sheet-forming peptides. Contrary to the regular beta-sheet structure of the amyloid fibril, the dimers exhibit all possible combinations of beta-sheets, with an overall preference for antiparallel arrangements. Through statistical analysis of 1,000 dimerization trajectories, each 1 ns in length, we have demonstrated that the observed distribution of dimer configurations is kinetically determined; hydrophobic interactions orient the peptides so as to minimize the solvent accessible surface area, and the dimer structures become trapped in energetically unfavorable conformations. Once the hydrophobic contacts are present, the backbone hydrogen bonds form rapidly by a zipper-like mechanism. The initial nonequilibrium structures formed are stable during the 1-ns simulation time for all five peptides at room temperature. In contrast, at higher temperatures, where rapid equilibration among different configurations occurs, the distribution follows the global energies. The relaxation time of dimers at room temperature was estimated to be longer than the time for diffusional encounters with other oligomers at typical concentrations. These results suggest that kinetic trapping could play a role in the structural evolution of early aggregates in amyloid fibrillogenesis.

Amyloid beta-Peptides↗
PubMed2005

Structure of dimeric mitochondrial ATP synthase: novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis.

The F1F0-ATP synthase exists as a dimer in mitochondria, where it is essential for the biogenesis of the inner membrane cristae. How two ATP synthase complexes dimerize to promote cristae formation is unknown. Here we resolved the structure of the dimeric F1F0 ATP synthase complex isolated from bovine heart mitochondria by transmission electron microscopy. The structure of the ATP synthase dimer has an overall conic appearance that is consistent with the proposed role of the dimeric enzyme in mitochondrial cristae biogenesis. The ATP synthase dimer interface is formed by contacts on both the F0 and F1 domains. A cross-bridging protein density was resolved which connects the two F0 domains on the intermembrane space side of the membrane. On the matrix side of the complex, the two F1 moieties are connected by a protein bridge, which is attributable to the IF1 inhibitor protein.

Animals↗
PubMed2001

Dimer formation drives the activation of the cell death protease caspase 9.

A critical step in the induction of apoptosis is the activation of the apoptotic initiator caspase 9. We show that at its normal physiological concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At the high concentrations used for crystal formation, caspase 9 is dimeric, and the structure reveals two very different active-site conformations within each dimer. One site closely resembles the catalytically competent sites of other caspases, whereas in the second, expulsion of the "activation loop" disrupts the catalytic machinery. We propose that the inactive domain resembles monomeric caspase 9. Activation is induced by dimerization, with interactions at the dimer interface promoting reorientation of the activation loop. These observations support a model in which recruitment by Apaf-1 creates high local concentrations of caspase 9 to provide a pathway for dimer-induced activation.

Apoptosis↗
PubMed1982

Recombinational bypass of pyrimidine dimers promoted by the recA protein of Escherichia coli.

recA protein, in the presence of single-stranded DNA binding protein and ATP, promotes the complete exchange of strands between circular single-stranded DNA containing pyrimidine dimers and a homologous linear duplex, converting the pyrimidine dimer-containing single-stranded DNA to a circular duplex. Bypass of a pyrimidine dimer during the branch-migration phase of the reaction requires approximately 20 seconds, a rate 1/50th of that in the absence of the dimer. The circular duplex product is specifically incised by the pyrimidine dimer-specific T4 endonuclease V, and the resulting 3' hydroxyl termini can serve as primers for deoxynucleotide polymerization by DNA polymerase I. These findings indicate that recA protein serves a direct role in recombinational repair and demonstrate that the pyrimidine dimers that have been bypassed can be processed by enzymes of the excision-repair pathway.

Adenosine Triphosphate↗
PubMed1997

Buried asparagines determine the dimerization specificities of leucine zipper mutants.

Regulation of gene expression by many transcription factors is controlled by specific combinations of homo- and heterodimers through a short alpha-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at the a, d, e, and g positions of the (abcdefg)n heptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at four a positions in the dimer interface, using a genetic test for the specificity of dimer formation by lambda repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C/EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at the a positions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization.

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS8(-)9(-) and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8(-)9(-) in 293 cell line resulted in generation of iNOS8(-)9(-) mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8(-)9(-) did not form dimers. Similar to iNOSFL, iNOS8(-)9(-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two "control" iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.

Alternative Splicing↗
PubMed1998

Human erythropoietin dimers with markedly enhanced in vivo activity.

Human erythropoietin, a widely used and important therapeutic glycoprotein, has a relatively short plasma half-life due to clearance by glomerular filtration as well as by other mechanisms. We hypothesized that an erythropoietin species with a larger molecular size would exhibit an increased plasma half-life and, potentially, an enhanced biological activity. We now report the production of biologically active erythropoietin dimers and trimers by chemical crosslinking of the conventional monomeric form. We imparted free sulfhydryl residues to a pool of erythropoietin monomer by chemical modification. A second pool was reacted with another modifying reagent to yield monomer with maleimido groups. Upon mixing these two pools, covalently linked dimers and trimers were formed that were biologically active in vitro. The plasma half-life of erythropoietin dimers in rabbits was >24 h compared with 4 h for the monomers. Importantly, erythropoietin dimers were biologically active in vivo as shown by their ability to increase the hematocrits of mice when injected subcutaneously. In addition, the dimers exhibited >26-fold higher activity in vivo than did the monomers and were very effective after only one dose. Dimeric and other oligomeric forms of Epo may have an important role in therapy.

Animals↗
PubMed1998

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis.

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal ("tail") domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains ("heads") that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Dimerization↗
PubMed1997

Effect of dimerization on signal transduction and biological function of oncogenic Ros, insulin, and insulin-like growth factor I receptors.

The avian sarcoma virus UR2 codes for an oncogenic Gag-Ros fusion protein-tyrosine kinase (PTK). We have previously derived two retroviruses, T6 and NM1, coding for oncogenic Gag-insulin receptor and Gag-insulin-like growth factor I receptor (IGFR) fusion proteins, respectively. The Gag-IGFR fusion protein dimerizes, whereas Gag-Ros does not. To identify sequences affecting dimerization and the effect of dimerization on signaling and biological functions, we generated recombinants exchanging the extracellular and transmembrane sequences among the three fusion receptors. The presence of multiple cysteines in the Gag sequence appears to preclude dimerization, since deletion of the 3' cysteine residue allows for dimerization. Most of the chimeric receptors retain high PTK activity and induce transformation regardless of their configuration on the cell surface. UT, a UR2/T6 chimera, retained mitogenic activity but has a markedly reduced transforming ability, while UN7, a UR2/NM1 recombinant, which also harbors Y950F and F951S mutations in IGFR, exhibits dramatic reductions in both activities. All of the fusion receptors can phosphorylate insulin receptor substrate 1 and activate PI 3-kinase. UT protein induces Shc phosphorylation, whereas UN7 protein does not, but both are unable to activate mitogen-activated protein kinase. Our results show that overexpressed oncogenic Gag-fusion receptors do not require dimerization for their signaling and transforming functions and that the extracellular and transmembrane sequences of a receptor PTK can affect its specific substrate interactions.

Adaptor Proteins, Signal Transducing↗