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PubMed2003

The Neisseria meningitidis adhesion regulatory protein CrgA acts through oligomerization and interaction with RNA polymerase.

CrgA is a LysR-type transcriptional regulator involved in the intimate adhesion of Neisseria meningitidis to target human epithelial cells. It is negatively autoregulated, and its expression is transiently induced upon contact with target cells. We analysed the functional organization of CrgA using in frame deleted proteins. Four truncated proteins were constructed and purified. They were deleted between residues 20 and 40, 121 and 154, 111 and 181 and 268 and 291. Meningococcal mutants harbouring the corresponding deleted crgA alleles were also constructed. All mutants showed a reduced ability to adhere to epithelial cells. beta-Galactosidase activity assays using a crgA-lacZ transcriptional fusion showed that all the mutations except the 268-291 deletion resulted in loss of induction upon contact with target cells. Gel mobility shift assays and cross-linking assays showed that the oligomerization of CrgA is required for DNA binding and that the N-terminal part of CrgA is directly involved in DNA binding through a helix-turn-helix motif. The C-terminal region is also involved in DNA binding, probably by permitting the oligomerization of CrgA. The C-terminal region also seemed to interact with RNA polymerase. Therefore, the binding of CrgA and its interaction with RNA polymerase may inhibit the clearance of meningococcal promoters that are repressed by CrgA during the intimate adhesion of N. meningitidis to target cells.

Bacterial Adhesion↗
PubMed2003

DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids.

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.

Adenosine Triphosphate↗
PubMed2002

The evolution of monomeric and oligomeric betagamma-type crystallins. Facts and hypotheses.

The case of homologous monomeric gamma-type and oligomeric beta-type crystallins has been described and analyzed in evolutionary terms. Data and hypotheses from molecular genetics and structural investigations converge and suggest a novel three-phase model for the evolutionary history of crystallin-type proteins. In the divergent cascades of monomeric and oligomeric crystallins, a pivotal role was played by alterations in the gene segments encoding the C-terminal extensions and the intermotif or interdomain linker peptides. These were genomic hot spots where evolution experimented to produce the modern variety of betagamma-crystallin-type quaternary structures.

Animals↗
PubMed2001

Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies.

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV-1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV-1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti-(HIV-1) mAbs.

Antibodies, Monoclonal↗
PubMed2001

Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants.

The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 degrees C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.

Enzyme Stability↗
PubMed2003

Functional consequences of homo- but not hetero-oligomerization between transporters for the biogenic amine neurotransmitters.

Before this study, the human norepinephrine transporter (hNET) was the only member of the biogenic amine neurotransmitter transporter family that had not been demonstrated to be a functional homo-oligomer. Here, using two forms of the transporter, I155C and hNET-myc, with distinct antigenicity and inhibitor sensitivity, we demonstrated that hNET exists as a homo-oligomer. hNET I155C is a functional mutant and is sensitive to inactivation by the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate, while hNET-myc is resistant to inactivation by this reagent. Coimmunoprecipitation of these two forms demonstrated that a physical interaction exists between norepinephrine transporter monomers. Further characterization of this physical interaction has revealed that the activity of norepinephrine transporters depends on interactions between monomers. Because norepinephrine transporters and serotonin transporters are the only two members of the neurotransmitter transporter family endogenously expressed in the cell membrane of the same cells, placental syncytiotrophoblasts, we tested the ability of norepinephrine transporters and serotonin transporters to associate and function in a hetero-oligomeric form. Similarly, coexpression of hNET-myc with serotonin transporter-FLAG showed a physical interaction in coimmunoprecipitation assays. However, coexpression of serotonin and norepinephrine transporters did not sensitize norepinephrine transporter activity to inhibition by citalopram, a selective serotonin transport inhibitor. Thus, the norepinephrine transporter-serotonin transporter physical association did not produce functional consequences. Based on this, we propose that the transporters for biogenic amine neurotransmitters interact functionally in homo- but not hetero-oligomeric forms.

Amino Acid Sequence↗
PubMed2004

Oligomerization and neurotoxicity of the amyloid ADan peptide implicated in familial Danish dementia.

Familial Danish dementia (FDD) is a rare neurodegenerative disorder, which is pathologically characterized by widespread cerebral amyloid angiopathy, parenchymal protein deposits and neurofibrillary degeneration. FDD is associated with mutation in the BRI gene. In FDD a decamer duplication between codons 265 and 266 in the 3' region of the BRI gene originates an amyloid peptide named ADan, 11 residues longer than the wild-type peptide produced from the normal BRI gene. ADan deposits have been found widely distributed in the CNS of FDD cases. The deposits of ADan are predominantly non-fibrillar aggregates. We show here that synthetic ADan forms oligomers in vitro, seen by Tricine-PAGE and gel filtration, and higher aggregates, which are seen by atomic force spectroscopy and electron microscopy as carrot-shaped objects that bunch together. Here we report that oligomeric ADan is toxic to neuronal cell lines. We find that the soluble non-fibrillar oligomeric species of both the reduced and oxidized forms of ADan are toxic. These results support the idea that the non-fibrillar soluble aggregates are the pathogenic species, which may play a central role in the pathogenesis of FDD, and imply that similar mechanism may also be involved in other neurodegenerative diseases associated with amyloid deposits.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Transforming growth factor-beta(1) induces apoptosis in rat FaO hepatoma cells via cytochrome c release and oligomerization of Apaf-1 to form a approximately 700-kd apoptosome caspase-processing complex.

In mammalian cells, non receptor-mediated apoptosis occurs via the cytochrome c-dependent assembly of a approximately 700-kd apoptotic protease-activating factor 1 (Apaf-1)/caspase-9 containing apoptosome complex. This initiates the postmitochondrial-mediated effector caspase cascade. We now show that receptor mediated transforming growth factor beta(1) (TGF-beta(1))-induced apoptosis in rat hepatoma cells is accompanied by processing and activation of caspases-2, -3, -7, and -8. Furthermore, we show that caspase activation is mediated via the release of cytochrome c and the oligomerization of Apaf-1 into an approximately 700-kd apoptosome complex. Similarly, in vitro activation of hepatoma cell lysates with 2'-deoxyadenosine 5'-triphosphate (dATP) results in the formation of the approximately 700-kd apoptosome complex, which recruits and processes caspases-3 and -7. Z-VAD.FMK [benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone], the pan-caspase inhibitor totally inhibits dATP-stimulated caspase activation but does not block the assembly of the large Apaf-1 containing apoptosome complex. However, the recruitment and subsequent processing of caspases-3 and -7 to the apoptosome is blocked. Similarly, in intact cells, although Z-VAD.FMK blocked TGF-beta(1)-induced apoptosis, it did not prevent the oligomerization of Apaf-1 into the apoptosome. However, recruitment and processing of caspases-3 and -7 were prevented by Z-VAD.FMK. These data show that TGF-beta(1) induces apoptosis via release of cytochrome c and activation of the Apaf-1 apoptosome complex, which initiates the caspase cascade.

Animals↗
PubMed2004

In silico study of amyloid beta-protein folding and oligomerization.

Experimental findings suggest that oligomeric forms of the amyloid beta protein (Abeta) play a critical role in Alzheimer's disease. Thus, elucidating their structure and the mechanisms of their formation is critical for developing therapeutic agents. We use discrete molecular dynamics simulations and a four-bead protein model to study oligomerization of two predominant alloforms, Abeta40 and Abeta42, at the atomic level. The four-bead model incorporates backbone hydrogen-bond interactions and amino acid-specific interactions mediated through hydrophobic and hydrophilic elements of the side chains. During the simulations we observe monomer folding and aggregation of monomers into oligomers of variable sizes. Abeta40 forms significantly more dimers than Abeta42, whereas pentamers are significantly more abundant in Abeta42 relative to Abeta40. Structure analysis reveals a turn centered at Gly-37-Gly-38 that is present in a folded Abeta42 monomer but not in a folded Abeta40 monomer and is associated with the first contacts that form during monomer folding. Our results suggest that this turn plays an important role in Abeta42 pentamer formation. Abeta pentamers have a globular structure comprising hydrophobic residues within the pentamer's core and hydrophilic N-terminal residues at the surface of the pentamer. The N termini of Abeta40 pentamers are more spatially restricted than Abeta42 pentamers. Abeta40 pentamers form a beta-strand structure involving Ala-2-Phe-4, which is absent in Abeta42 pentamers. These structural differences imply a different degree of hydrophobic core exposure between pentamers of the two alloforms, with the hydrophobic core of the Abeta42 pentamer being more exposed and thus more prone to form larger oligomers.

Amyloid beta-Peptides↗
PubMed2005

Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor.

S100B protein is elevated in the brains of patients with early stages of Alzheimer's disease and Down's syndrome. S100A4 is correlated with the development of metastasis. Both proteins bind to p53 tumor suppressor. We found that both S100B and S100A4 bind to the tetramerization domain of p53 (residues 325-355) only when exposed in lower oligomerization states and so they disrupt the tetramerization of p53. In addition, S100B binds to the negative regulatory and nuclear localization domains, which results in a very tight binding to p53 protein sequences that exposed the tetramerization domain in their C terminus. Because the trafficking of p53 depends on its oligomerization state, we suggest that S100B and S100A4 could regulate the subcellular localization of p53. But, the differences in the way these proteins bind to p53 could result in S100B and S1004 having different effects on p53 function in cell-cycle control.

Amino Acid Sequence↗
PubMed2006

Membrane-dependent oligomeric structure and pore formation of a beta-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR.

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a beta-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE/POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)/cholesterol bilayers]. (1)H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC/cholesterol bilayer. (19)F spin diffusion data indicates that 75% of the beta-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN)(n) multimer suggests a membrane-inserted beta-barrel enclosing a water pore. The lipids surrounding the beta-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC/cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of beta-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.

Anions↗
PubMed2006

Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis.

Phototaxis allows archaea to adjust flagellar motion in response to light. In the photophobic response of Natronobacterium pharaonis, light-activated sensory rhodopsin II causes conformational changes in the transducer II protein (pHtrII), initiating the two-component signaling system analogous to bacterial chemotaxis. pHtrII's cytoplasmic domain (pHtrII-cyt) is homologous to the cytoplasmic domains of eubacterial chemotaxis receptors. Chemotaxis receptors require dimerization for activity and are in vivo-organized in large clusters. In this study we investigated the oligomerization and aggregation states of pHtrII-cyt by using chemical cross-linking, analytical gel-filtration chromatography, and small-angle neutron scattering. We show that pHtrII-cyt is monomeric in dilute buffers, but forms dimers in 4 M KCl, the physiological salt concentration for halophilic archaea. At high ammonium sulfate concentration, the protein forms higher-order aggregates. The monomeric protein has a rod-like shape, 202 A in length and 14.4 A in diameter; upon dimerization the length increases to 248 A and the diameter to 18.2 A. These results suggest that under high salt concentration the shape and oligomerization state of pHtrII-cyt are comparable to those of chemotaxis receptors.

Archaeal Proteins↗
PubMed2002

Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen.

Assembly of anthrax toxin complexes at the mammalian cell surface involves competitive binding of the edema factor (EF) and lethal factor (LF) to heptameric oligomers and lower order intermediates of PA(63), the activated carboxyl-terminal 63-kDa fragment of protective antigen (PA). We used sequence differences between PA(63) and homologous PA-like proteins to delineate a region within domain 1' of PA that may represent the binding site for these ligands. Substitution of alanine for any of seven residues in or near this region (R178, K197, R200, P205, I207, I210, and K214) strongly inhibited ligand binding. Selected mutations from this set were introduced into two oligomerization-deficient PA mutants, and the mutants were used in various combinations to map the single ligand site within dimeric PA(63). The site was found to span the interface between two adjacent subunits, explaining the dependence of ligand binding on PA oligomerization. The locations of residues comprising the site suggest that a single ligand molecule sterically occludes two adjacent sites, consistent with the finding that the PA(63) heptamer binds a maximum of three ligand molecules. These results elucidate the process by which the components of anthrax toxin, and perhaps other binary bacterial toxins, assemble into toxic complexes.

Adenylyl Cyclases↗
PubMed2003

Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss.

A molecular basis for memory failure in Alzheimer's disease (AD) has been recently hypothesized, in which a significant role is attributed to small, soluble oligomers of amyloid beta-peptide (A beta). A beta oligomeric ligands (also known as ADDLs) are known to be potent inhibitors of hippocampal long-term potentiation, which is a paradigm for synaptic plasticity, and have been linked to synapse loss and reversible memory failure in transgenic mouse AD models. If such oligomers were to build up in human brain, their neurological impact could provide the missing link that accounts for the poor correlation between AD dementia and amyloid plaques. This article, using antibodies raised against synthetic A beta oligomers, verifies the predicted accumulation of soluble oligomers in AD frontal cortex. Oligomers in AD reach levels up to 70-fold over control brains. Brain-derived and synthetic oligomers show structural equivalence with respect to mass, isoelectric point, and recognition by conformation-sensitive antibodies. Both oligomers, moreover, exhibit the same striking patterns of attachment to cultured hippocampal neurons, binding on dendrite surfaces in small clusters with ligand-like specificity. Binding assays using solubilized membranes show oligomers to be high-affinity ligands for a small number of nonabundant proteins. Current results confirm the prediction that soluble oligomeric A beta ligands are intrinsic to AD pathology, and validate their use in new approaches to therapeutic AD drugs and vaccines.

Alzheimer Disease↗
PubMed2002

Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways.

Amyloid beta-protein (Abeta) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of Abeta in AD is new evidence that oligomeric forms of Abeta are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Abeta, Abeta42, with the disease. Detailed knowledge of the structure and assembly dynamics of Abeta thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Abeta oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Abeta40 and Abeta42. Carefully prepared aggregate-free Abeta40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Abeta42 preferentially formed pentamerhexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Abeta40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Abeta42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Abeta42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

Alzheimer Disease↗
PubMed2000

Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: functional complementation of specific defects in gp120 and gp41.

The envelope glycoprotein (Env) of HIV-1 is displayed on the surface of the virion or infected cell as an oligomer of multiple gp120/gp41 complexes. We sought to unravel the relationships between this oligomeric structure and the requirements for sequential interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a quantitative cell fusion assay to examine the effects of coexpressing pairs of Envs, each nonfunctional because of a specific defect in one of the essential properties. We observed efficient fusion activity upon coexpression of two Env variants, one containing a gp41 subunit with a mutated fusion peptide and the other containing a gp120 subunit with a mutated CD4 binding site or a mismatched coreceptor specificity. We also observed fusion upon coexpression of two Env variants with distinct gp120 defects, i.e., a CD4 binding site mutation and the incorrect coreceptor specificity determinants. Coimmunoprecipitation experiments verified the efficient formation of mixed oligomers, suggesting that the observed fusion reflected subunit complementation within the oligomeric complex. These results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. The implications of these findings for Env function and virus neutralization are discussed.

3T3 Cells↗
PubMed2003

In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding.

The matrix protein VP40 plays a critical role in Ebola virus assembly and budding, a process that utilizes specialized membrane domains known as lipid rafts. Previous studies with purified protein suggest a role for oligomerization of VP40 in this process. Here, we demonstrate VP40 oligomers in lipid rafts of mammalian cells, virus-like particles, and in the authentic Ebola virus. By mutagenesis, we identify several critical C-terminal sequences that regulate oligomerization at the plasma membrane, association with detergent-resistant membranes, and vesicular release of VP40, directly linking these phenomena. Furthermore, we demonstrate the active recruitment of TSG101 into lipid rafts by VP40. We also report the successful application of the biarsenic fluorophore, FlAsH, combined with a tetracysteine tag for imaging of Ebola VP40 in live cells.

Cell Line↗
PubMed1972

Enzymatic oligomerization of bacteriophage P22 DNA and of linear Simian virus 40 DNA.

Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in the presence of polynucleotide ligase coded for by the coliphage T4. The reaction has been monitored by sucrose density-gradient centrifugation and electron microscopy. It goes slowly and gives yields of 30-40%. The products are mainly dimers and trimers, but higher oligomers are also present.DNA ligase extracted from uninfected Escherichia coli seems unable to perform a similar reaction, which is concluded to involve the fully base-paired termini. Linear double-stranded molecules of simian virus(SV) 40 DNA, produced by the action of the bacterial restriction endonuclease R(1), are oligomerized by either ligase; therefore, this reaction seems to involve single-stranded cohesive ends. No mixed products could be found when P22 DNA and linear SV 40 DNA were exposed together to the T4 ligase.

Carbon Isotopes↗