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PubMed1994

Functional oligomerization of purified human papillomavirus types 16 and 6b E7 proteins expressed in Escherichia coli.

Purified non-fused soluble human papillomavirus type 16 and 6b E7 proteins expressed in Escherichia coli were found to form oligomers. For both proteins, several degrees of oligomerization were demonstrated by gel filtration, dynamic laser light scattering and scanning electron microscopy. Oligomerization was dependent on the concentration of E7 protein. Oligomerized E7 proteins were able to bind the retinoblastoma gene product pRB and stimulated DNA synthesis when introduced into cells.

Base Sequence↗
PubMed1998

Multiple oligomeric forms of glucose-6-phosphate dehydrogenase in cyanobacteria and the role of OpcA in the assembly process.

Multiple molecular forms of glucose-6-phosphate dehydrogenase (G6PDH) were detected by activity staining in non-denaturing polyacrylamide gels of cell-free extracts from a range of cyanobacteria including Anabaena sp. PCC 7120, Synechococcus sp. PCC 7942, Plectonema boryanum PCC 73110, Synechocystis sp. PCC 6803, Nostoc sp. MAC PCC 8009 and the marine strain Synechococcus sp. WH7803. In most of the species tested, the profile of G6PDH activities was modulated by the growth of the cells in the presence of exogenous 10 mM glucose. Using an antiserum raised against a fragment of G6PDH from Anabaena sp. PCC 7120, it was shown that the different molecular forms of G6PDH all contained an antigenically related subunit, suggesting that the different forms arose from different quaternary structures involving the same monomer. An insertion mutant of Synechococcus sp. PCC 7942 was constructed in which the opcA gene, adjacent to zwf (encoding G6PDH), was disrupted. Although no reduction in the amount of G6PDH monomers (Zwf) was observed in the opcA mutant, activity staining of native gels indicated that most of this protein is not assembled into one of the active oligomeric forms. The oligomerization of G6PDH in extracts of the opcA mutant was stimulated in vitro by a factor present in crude extracts of the wild-type, suggesting that the product of the opcA gene is involved in the oligomerization and activation of G6PDH.

Bacterial Proteins↗
PubMed2003

Transmissible Burkholderia cepacia genomovar IIIa strains bind and convert monomeric iron(III) protoporphyrin IX into the mu-oxo oligomeric form.

Burkholderia cepacia isolates of genomovar III are highly transmissible amongst patients with cystic fibrosis (CF) and express a 97 kDa putative haem-binding protein (HBP) [Smalley, J. W., Charalabous, P., Birss, A. J. & Hart, C. A. (2001). Clin Diagn Lab Immunol 8, 509-514]. An investigation of the interactions of iron(III) protoporphyrin IX with epidemic and non-epidemic strains of B. cepacia to determine the role of the above protein in haem acquisition and binding is reported herein. Spectrophotometric titrations of cell suspensions of genomovar IIIa strains BC7 and C5424 with iron(III) protoporphyrin IX, at pH 7.0, resulted in the depletion of Fe(III)PPIX.OH monomers and formation of the micro -oxo oligomeric species, [Fe(III)PPIX](2)O. Difference spectroscopy indicated a continuous conversion of the monomeric iron(III) protoporphyrin IX into micro -oxo oligomers. Incubations with Fe(III)PPIX.OH monomers at pH 6.5 also showed that cells could shift the equilibrium to generate the micro -oxo oligomeric form. Genomovar I strains ATCC 25416 and LMG 17997 were unable to mediate this conversion. SDS-PAGE of genomovar IIIa strains exposed to Fe(III)PPIX.OH at pH 6.5 followed by tetramethylbenzidine/H(2)O(2) staining revealed, in addition to the 97 kDa HBP, two proteins of 77 and 149 kDa located in the outer membrane which bound Fe(III)PPIX.OH monomers. These proteins were absent from the genomovar I strains. Genomovar IIIa strains BC7 and C5424 showed increased cellular binding of [Fe(III)PPIX](2)O, and as a consequence, displayed increased catalase activities compared to cells of the genomovar I isolates. It is concluded that, in addition to the putative 97 kDa HBP, B. cepacia genomovar IIIa strains express two outer-membrane proteins which function to bind and convert Fe(III)PPIX.OH monomers into the micro -oxo oligomeric form, [Fe(III)PPIX](2)O. The ability to perform this conversion at both neutral and slightly acidic pHs may enable epidemic strains to withstand attack from neutrophil-derived H(2)O(2) in the inflamed CF lung.

Bacterial Outer Membrane Proteins↗
PubMed2025

Oligomerization and positive feedback on membrane recruitment encode dynamically stable PAR-3 asymmetries in the C. elegans zygote.

Studies of PAR polarity have emphasized a paradigm in which mutually antagonistic PAR proteins form complementary polar domains in response to transient cues. A growing body of work suggests that the oligomeric scaffold PAR-3 can form unipolar asymmetries without mutual antagonism, but how it does so is largely unknown. Here we combine single molecule analysis and modeling to show how the interplay of two positive feedback loops promotes dynamically stable unipolar PAR-3 asymmetries in early C. elegans embryos. First, the intrinsic dynamics of PAR-3 membrane binding and oligomerization encode negative feedback on PAR-3 dissociation. Second, membrane-bound PAR-3 promotes its own recruitment through a mechanism that requires the anterior polarity proteins PAR-6 and PKC-3. Using a kinetic model tightly constrained by our experimental measurements, we show that these two feedback loops are individually required and jointly sufficient to encode dynamically stable and locally inducible unipolar PAR-3 asymmetries in the absence of posterior inhibition. Given the central role of PAR-3, and the conservation of PAR-3 membrane-binding, oligomerization, and core interactions with PAR-6/PKC-3, these results have widespread implications for PAR-mediated polarity in metazoa.

Journal Article↗
PubMed1991

TFEB has DNA-binding and oligomerization properties of a unique helix-loop-helix/leucine-zipper family.

The DNA-binding factor TFEB contains adjacent helix-loop-helix (HLH) and leucine zipper (LZ) domains flanked by an upstream basic region. This arrangement of interactive motifs has recently been observed in several other transcription factors and in the Myc family of oncogenes. TFEB was isolated by virtue of its binding to the major late promoter of adenovirus. DNA binding by a soluble protein was achieved by deleting a hydrophobic amino-terminal domain and permitted the structural analysis of the oligomerization and DNA-binding properties of TFEB. TFEB specifically bound DNA as both a homodimer and a heterodimer with another b-HLH-LZ protein TFE3. The LZ domain was essential for homo- or hetero-oligomerization and high-affinity DNA binding. In the absence of DNA a tetramer-sized form of TFEB was observed that dissociates to bind added DNA as a dimer. Binding by TFEB and TFE3 to related, but different, naturally occurring DNA target sequences was observed with distinct binding preferences. Analysis of basic domain residues in this family of proteins revealed a pattern of sequence conservation predictive of an interacting alpha-helical face. Common oligomerization and DNA-binding features suggest the b-HLH-LZ domain structure to define a distinct family of DNA-binding factors.

Adenoviridae↗
PubMed2004

Structure and mutational analysis of a plant mitochondrial nucleoside diphosphate kinase. Identification of residues involved in serine phosphorylation and oligomerization.

We report the first crystal structure of a plant (Pisum sativum L. cv Oregon sugarpod) mitochondrial nucleoside diphosphate kinase. Similar to other eukaryotic nucleoside diphosphate kinases, the plant enzyme is a hexamer; the six monomers in the asymmetric unit are arranged as trimers of dimers. Different functions of the kinase have been correlated with the oligomeric structure and the phosphorylation of Ser residues. We show that the occurrence of Ser autophosphorylation depends on enzymatic activity. The mutation of the strictly conserved Ser-119 to Ala reduced the Ser phosphorylation to about one-half of that observed in wild type with only a modest change of enzyme activity. We also show that mutating another strictly conserved Ser, Ser-69, to Ala reduces the enzyme activity to 6% and 14% of wild-type using dCDP and dTDP as acceptors, respectively. Changes in the oligomerization pattern of the S69A mutant were observed by cross-linking experiments. A reduction in trimer formation and a change in the dimer interaction could be detected with a concomitant increase of tetramers. We conclude that the S69 mutant is involved in the stabilization of the oligomeric state of this plant nucleoside diphosphate kinase.

Amino Acid Sequence↗
PubMed2004

Oligomerization of hydrophobin SC3 in solution: from soluble state to self-assembly.

Hydrophobin SC3 is a protein with special self-association properties that differ depending on whether it is in solution, on an air/water interface or on a solid surface. Its self-association on an air/water interface and solid surface have been extensively characterized. The current study focuses on its self-association in water because this is the starting point for the other two association processes. Size-exclusion chromatography was used to fractionate soluble-state SC3. Real-time multiangular light scattering detection of the eluate indicated that SC3 mainly exists as a dimer in buffer, accompanied with a small amount of monomer, tetramer, and larger aggregates. Dimeric SC3 has very likely an elongated shape, as indicated by the hydrodynamic radius determined by using dynamic light scattering (DLS) and fluorescence anisotropy measurements on dansyl-labeled SC3. Size-exclusion chromatography experiments also indicated that the protein oligomerizes very slowly at low temperature (4 degrees C) but rather rapidly at room temperature. Ionic strength plays an important role in the oligomerization; a short-lived monomeric SC3 species could be observed in pure water. Oligomerization was not affected by low pH but was accelerated by high pH. Fluorescence resonance energy transfer showed that dissociation occurred when the protein concentration was lowered; a large population of oligomers, presumably dimers, dissociate when the protein concentration is <4.5 microg/mL. This value is similar to the critical concentration for SC3 self-assembly. Therefore, dimeric SC3 is indicated to be the building block for both aggregation in solution and self-assembly at hydrophobic/hydrophilic interfaces.

Chromatography, Gel↗
PubMed1999

Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.

Amino Acid Substitution↗
PubMed1999

Nucleotide-dependent oligomerization of ClpB from Escherichia coli.

Self-association of ClpB (a mixture of 95- and 80-kDa subunits) has been studied with gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. Monomeric ClpB predominates at low protein concentration (0.07 mg/mL), while an oligomeric form is highly populated at >4 mg/mL. The oligomer formation is enhanced in the presence of 2 mM ATP or adenosine 5'-O-thiotriphosphate (ATPgammaS). In contrast, 2 mM ADP inhibits full oligomerization of ClpB. The apparent size of the ATP- or ATPgammaS-induced oligomer, as determined by gel filtration, sedimentation velocity and electron microscopy image averaging, and the molecular weight, as determined by sedimentation equilibrium, are consistent with those of a ClpB hexamer. These results indicate that the oligomerization reactions of ClpB are similar to those of other Hsp100 proteins.

Adenosine Triphosphate↗
PubMed2006

The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA-binding activities on separate domains.

The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains.

Amino Acid Motifs↗
PubMed1990

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures.

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.

Chromatography, Gel↗
PubMed1992

A major oligomeric fibroblast proteoglycan identified as a novel large form of type-XII collagen.

Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600-kDa polypeptide is likely to be a dimer of two smaller subunits which are cross-linked by non-reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350-kDa and 600-kDa (but not the 300-kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S] sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non-collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type-XII collagen from its full-length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209-221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type-XII collagen: its subunits are larger by one third, and it is a proteoglycan.

Amino Acid Sequence↗
PubMed2007

The alpha1b-adrenoceptor exists as a higher-order oligomer: effective oligomerization is required for receptor maturation, surface delivery, and function.

Approaches to identify G protein-coupled receptor oligomers rather than dimers have been lacking. Using concatamers of fluorescent proteins, we established conditions to monitor sequential three-color fluorescence resonance energy transfer (3-FRET) and used these to detect oligomeric complexes of the alpha(1b)-adrenoceptor in single living cells. Mutation of putative key hydrophobic residues in transmembrane domains I and IV resulted in substantial reduction of sequential 3-FRET and was associated with lack of protein maturation, prevention of plasma membrane delivery, and elimination of signaling function. Although these mutations prevented cell surface delivery, bimolecular fluorescence complementation studies indicated that they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I plus transmembrane domain IV mutated receptor. The transmembrane domain I plus transmembrane domain IV mutated receptor was a "dominant-negative" in blocking cell surface delivery of the wild-type receptor. Mutations only in transmembrane domain I did not result in a reduction in 3-FRET, whereas restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV, however, were sufficient to eliminate cell surface delivery. Terminal N-glycosylation is insufficient to determine cell surface delivery because both transmembrane domain I and transmembrane domain IV mutants matured as effectively as the wild-type receptor. These data indicate that the alpha(1b)-adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.

Cell Line↗
PubMed1999

Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin.

Katanin, a member of the AAA adenosine triphosphatase (ATPase) superfamily, uses nucleotide hydrolysis energy to sever and disassemble microtubules. Many AAA enzymes disassemble stable protein-protein complexes, but their mechanisms are not well understood. A fluorescence resonance energy transfer assay demonstrated that the p60 subunit of katanin oligomerized in an adenosine triphosphate (ATP)- and microtubule-dependent manner. Oligomerization increased the affinity of katanin for microtubules and stimulated its ATPase activity. After hydrolysis of ATP, microtubule-bound katanin oligomers disassembled microtubules and then dissociated into free katanin monomers. Coupling a nucleotide-dependent oligomerization cycle to the disassembly of a target protein complex may be a general feature of ATP-hydrolyzing AAA domains.

Adenosine Triphosphatases↗
PubMed2003

Activation of procaspases by FK506 binding protein-mediated oligomerization.

Oligomerization is an important biological mechanism for regulating signal transduction. Activation of caspases during apoptosis is triggered by adaptor protein-mediated oligomerization of initiator procaspases. To facilitate the study of initiator caspase activation, a system that allows inducible activation of various caspases both in vitro and in vivo is highly desired. Here we describe such a caspase activation system that is based on FK506 binding protein (FKBP)-mediated oligomerization. The NH(2)-terminal prodomains of initiator procaspases that facilitate the interaction between procaspases and their adaptor proteins are replaced by a derivative of FKBP called Fv. The Fv-caspase fusions can then be dimerized by a synthetic divalent Fv ligand, AP20187, which binds strongly to Fv but weakly to the endogenous FKBPs. This FKBP-based system may be widely applicable to the study of the regulation and functions of caspases.

Caspase 8↗
PubMed1987

Quantitation of monomeric and oligomeric forms of membrane-bound staphylococcal alpha-toxin by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody.

A murine monoclonal antibody generated against staphylococcal alpha-toxin was shown to react only with the monomeric (native), 3S form of the toxin. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) constructed with this antibody permitted detection of 0.25 to 0.5 ng of native toxin per ml. Toxin oligomers formed either by heat aggregation in solution, on target erythrocyte membranes, or on phosphatidylcholine-cholesterol liposomes were unreactive in the ELISA when membranes were solubilized with the nondenaturing detergent Triton X-100. After dissociation of the oligomers by boiling in sodium dodecyl sulfate, however, the ELISA reactivity of the liberated 3S toxin was fully restored. Parallel determinations of membrane-bound toxin with sodium dodecyl sulfate and Triton X-100 solubilization thus permitted direct quantitation of total and monomeric toxin, respectively; the difference between these two values was represented by toxin oligomers. The detection limits for membrane-bound oligomeric and monomeric toxin on erythrocyte membranes are in the order of 100 molecules and 1 molecule per cell, respectively. Using this ELISA, we show that over 90% of alpha-toxin molecules bound to target membranes at 37 degrees C are in oligomeric form. Evidence is given that the monoclonal antibody neutralizes alpha-toxin by inhibiting its binding to both rabbit and human erythrocytes. This ELISA is the first assay that quantitatively discriminates between mono- and oligomeric forms of a pore-forming protein on target cell membranes.

Antibodies, Monoclonal↗
PubMed1983

Absence of oligomeric murein intermediates in Escherichia coli.

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.

Escherichia coli↗
PubMed2000

Domain structure, oligomeric state, and mutational analysis of PpsR, the Rhodobacter sphaeroides repressor of photosystem gene expression.

The transcription factor PpsR from the facultative photoheterotroph Rhodobacter sphaeroides is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in ppsR. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action.

Amino Acid Sequence↗