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PubMed2005

The essential role of the flexible termini in the temperature-responsiveness of the oligomeric state and chaperone-like activity for the polydisperse small heat shock protein IbpB from Escherichia coli.

Small heat shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones that are believed to prevent irreversible aggregation of other cellular proteins under stress conditions. One of the most prominent features of sHSPs is that they exist as homo-oligomers. Examples of both monodisperse and polydisperse oligomers are found within this family. The small heat shock inclusion-body binding protein B (IbpB) of Escherichia coli, originally discovered as a component of inclusion bodies, exhibits a pronounced polydispersity in its oligomeric state. This research was performed to elucidate the temperature effect on the oligomeric state and chaperone-like activity of the polydisperse IbpB oligomers, as well as the structural basis for such a temperature effect. The data presented here demonstrate that the large oligomers of IbpB progressively dissociate into smaller ones at increasing heat-shock temperatures, accompanied by a notable enhancement of chaperone-like activities. The secondary structure, enriched mainly by beta-strands, is slightly changed with such temperature increases. The dimeric building blocks, which seem to be highly stable, act as the functional unit of IbpB. Limited proteolysis was used to identify the susceptible sites in IbpB that may compose the subunit interfaces, which indicated that the 11 residues at both the N and the C terminus are highly flexible and the removal of each will lead to the formation of dimers, as well as the disappearance of chaperone-like activities. Truncation of 11 residues from either end, using recombinant DNA technology, also led to the formation of dimeric mutant IbpB proteins lacking chaperone-like activities. Taken together, the flexible termini appear to be essential for small heat shock protein IbpB to generate various temperature-responsive oligomers, which exhibit various levels of chaperone-like activities, by interlinking or separating the dimer building blocks.

Circular Dichroism↗
PubMed2005

Environmental control of the in vivo oligomerization of nucleoid protein H-NS.

The nucleoid-associated transcriptional repressor H-NS forms both dimers and tetramers in vivo. Two types of two-hybrid systems, one capable of detecting protein dimerization and the other protein tetramerization, have been used to determine whether environmental changes could affect the oligomerization capacity of this protein in the cell. Increasing the temperature from 37 degrees C to 48 degrees C and changing the pH between 4.0 and 9.0 did not influence either dimerization or tetramerization, whereas lowering the temperature below 25 degrees C and increasing osmolarity were found to reduce the formation of H-NS tetramers, which are the active form of this protein, without affecting dimerization. These findings provide a rationale to explain the induction of H-NS expression during cold-shock, suggest a mechanism contributing to derepressing osmotic-shock genes transcriptionally regulated by H-NS and indicate that changes of the oligomerization properties of H-NS do not play a role in the H-NS and temperature-dependent control of virulence gene expression.

Bacterial Proteins↗
PubMed2006

Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry.

Numerous debilitating human disorders result from protein misfolding and amyloid formation. Despite the grave nature of these maladies, our understanding of the structural mechanism of fibril assembly is limited. Of paramount importance is the need to identify and characterize oligomeric species formed early during fibril assembly, so that the nature of the initiating assembly mechanism can be revealed and species that may be toxic to cells identified. However, the transient nature of early oligomeric species, combined with their heterogeneity and instability, has precluded detailed analysis to date. Here, we have used electrospray ionisation mass spectrometry (ESI-MS), complemented by analytical ultracentrifugation (AUC) and measurements of thioflavin-T fluorescence, to monitor the early stages of assembly of amyloid-like fibrils formed from human beta-2-microglobulin (beta2m) in vitro. We show that worm-like fibrils that form with nucleation-independent kinetics assemble by a mechanism consistent with monomer addition, with species ranging from monomer to > or = 13-mer being identified directly and uniquely as transient assembly intermediates. By contrast, only monomers, dimers, trimers and tetramers are observed during nucleated growth, which leads to the formation of long straight fibrils. The results highlight the unique power of non-covalent ESI-MS to identify protein assembly intermediates in complex heterogeneous systems and demonstrate its great potential to identify and characterise individual species formed early during amyloid assembly.

Alzheimer Disease↗
PubMed2006

The NMR structure of the gpU tail-terminator protein from bacteriophage lambda: identification of sites contributing to Mg(II)-mediated oligomerization and biological function.

During the late stages of lambda bacteriophage assembly, the protein gpU terminates tail polymerization and participates at the interface between the mature capsid and tail components. When it engages the lambda tail, gpU undergoes a monomer-hexamer transition to achieve its biologically active form. Towards understanding how gpU participates in multiple protein-protein interactions, we have solved the structure of gpU in its monomeric state using NMR methods. The structure reveals a mixed alpha/beta motif with several dynamic loops at the periphery. Addition of 20 mM MgCl(2) is known to oligomerize gpU in the absence of its protein partners. Multiple image analysis of electron micrographs revealed ring-like structures of magnesium ion saturated gpU with a 30 A pore, consistent with its function as a portal for the passage of viral DNA into the host bacterium. The ability of magnesium ions to promote oligomerization was lost when substitutions were made at a cluster of acidic amino acids in the vicinity of helix alpha2 and the beta1-beta2 loop. Furthermore, substitutions at these sites abolished the biological activity of gpU.

Amino Acid Sequence↗
PubMed2006

The three-dimensional structure of an eukaryotic glutamine synthetase: functional implications of its oligomeric structure.

The structure of the prokaryotic glutamine synthetases type I (GS-I), key enzymes in nitrogen metabolism, was determined several years ago by X-ray diffraction, and consists of a double hexameric ring. The structure of the eukaryotic GS from the plant Phaseolus vulgaris (Glutamine synthetase type II; GS-II) has now been determined at low-resolution using electron microscopy and image processing, and consists of an octamer composed of two tetramers placed back-to-back and rotated 90 degrees with respect to each other. The oligomeric structure possesses a twofold symmetry, very suggestive of each tetramer being composed of two dimers. This is reinforced by the fact that dimers are isolated as a stable albeit non-functional species during the purification procedure. Given the fact that the active site of all types of GS is formed by highly conserved residues located in the interface of two interacting monomers, the geometry of the reconstructed tetramer suggests that it only contains two functional active sites, i.e., an active site per dimer. This is supported by biochemical data, which reveal that while the octamer binds eight ATP molecules, it only binds four molecules of the transition state analogue and GS inhibitor methionine-(S)-sulfoximine-P (MetSox-P). All this suggests for the GS-II enzyme an oligomeric structure containing four active sites and four possible regulatory sites, which might point to a complex regulatory behavior.

Amino Acid Sequence↗
PubMed2005

ApoE isoform-specific effects on LTP: blockade by oligomeric amyloid-beta1-42.

Amyloid-beta1-42 (Abeta1-42) is crucial to Alzheimer disease (AD) pathogenesis but the conformation of the toxic Abeta species remains uncertain. AD risk is increased by apolipoprotein E4 (apoE4) and decreased by apoE2 compared with the apoE3 isoform, but whether inheritance of apoE4 represents a gain of negative or a loss of protective function is also unresolved. Using hippocampal slices from apoE knockout (apoE-KO) and human apoE2, E3, and E4 targeted replacement (apoE-TR) mice, we found that oligomeric Abeta1-42 inhibited long-term potentiation (LTP) with a hierarchy of susceptibility mirroring clinical AD risk (apoE4-TR > apoE3-TR = apoE-KO > apoE2-TR), and that comparable doses of unaggregated Abeta1-42 did not affect LTP. These data provide a novel link among apoE isoform, Abeta1-42, and a functional cellular model of memory. In this model, apoE4 confers a gain of negative function synergistic with Abeta1-42, apoE2 is protective, and the apoE-Abeta interaction is specific to oligomeric Abeta1-42.

Alzheimer Disease↗
PubMed2005

Differential effects of oligomeric and fibrillar amyloid-beta 1-42 on astrocyte-mediated inflammation.

Activated glia, as a result of chronic inflammation, are associated with amyloid-beta peptide (Abeta) deposits in the brain of Alzheimer's disease (AD) patients. In vitro, glia are activated by Abeta inducing secretion of pro-inflammatory molecules. Recent studies have focused on soluble oligomers (or protofibrils) of Abeta as the toxic species in AD. In the present study, using rat astrocyte cultures, oligomeric Abeta induced initial high levels of IL-1beta decreasing over time and, in contrast, fibrillar Abeta increased IL-1beta levels over time. In addition, oligomeric Abeta, but not fibrillar Abeta, induced high levels of iNOS, NO, and TNF-alpha. Our results suggest that oligomers induced a profound, early inflammatory response, whereas fibrillar Abeta showed less increase of pro-inflammatory molecules, consistent with a more chronic form of inflammation.

Alzheimer Disease↗
PubMed2005

Expression and characterization of disulfide bond use of oligomerized A2-Pancortins: extracellular matrix constituents in the developing brain.

The region-specific characteristics of the extracellular matrix are crucial for diverse functions in the brain. Pancortins/neuron-specific olfactomedin-related glycoproteins are components of the extracellular matrix. They comprise four alternatively spliced variants, Pancortin-1 to -4, which share a common portion, the B part, in the middle of their structure, have two pairs of alternatively spliced 5' regions, A1 and A2, and 3' regions, C1 and C2. Here we demonstrate that in mice, Pancortin-3 (A2-B-C1) and Pancortin-4 (A2-B-C2), which we have grouped together the A2-Pancortins, were transcribed early during the development of the brain in a region specific manner and were expressed very stably in vivo. They are N-glycosylated and secreted. Furthermore, we examined their ontogenetical expression profiles in the developing thalamus using antiserum against the common B region, since transient expressions of their mRNAs were notable there. In the developing thalami, they lasted long in oligomerized form even after the transcription of their mRNAs decreased to an undetectable level. Further analyses revealed that cysteine residues that are located in the common B part are important for homo- and hetero-oligomer formation of A2-Pancortins. When we substituted cysteine residues 45 and 47 with serine residues in that common B part, oligomerization of the A2-Pancortins was highly disturbed.

Age Factors↗
PubMed2006

Pathways and control of connexin oligomerization.

Connexins form gap junction channels that link neighboring cells into an intercellular communication network. Many cells that express multiple connexins produce heteromeric channels containing at least two connexins, which provides a means to fine tune gap junctional communication. Formation of channels by multiple connexins is controlled at two levels: by inherent structural compatibilities that enable connexins to hetero-oligomerize and by cellular mechanisms that restrict the formation of heteromers by otherwise compatible connexins. Here, I discuss roles for secretory compartments beyond the endoplasmic reticulum in connexin oligomerization and evidence that suggests that membrane microdomains help regulate connexin trafficking and assembly.

Animals↗
PubMed2005

Homo-oligomerization facilitates the interferon-antagonist activity of the ebolavirus VP35 protein.

We have identified a putative coiled-coil motif within the amino-terminal half of the ebolavirus VP35 protein. Cross-linking studies demonstrated the ability of VP35 to form trimers, consistent with the presence of a functional coiled-coil motif. VP35 mutants lacking the coiled-coil motif or possessing a mutation designed to disrupt coiled-coil function were defective in oligomerization, as deduced by co-immunoprecipitation studies. VP35 inhibits signaling that activates interferon regulatory factor 3 (IRF-3) and inhibits (IFN)-alpha/beta production. Experiments comparing the ability of VP35 mutants to block IFN responses demonstrated that the VP35 amino-terminus, which retains the putative coiled-coil motif, was unable to inhibit IFN responses, whereas the VP35 carboxy-terminus weakly inhibited the activation of IFN responses. IFN-antagonist function was restored when a heterologous trimerization motif was fused to the carboxy-terminal half of VP35, suggesting that an oligomerization function at the amino-terminus facilitates an "IFN-antagonist" function exerted by the carboxy-terminal half of VP35.

Amino Acid Motifs↗
PubMed2006

Oligomeric structure of the alpha1b-adrenoceptor: comparisons with rhodopsin.

The structural basis of the quaternary organization of rhodopsin has recently been explored and modeled. Because information obtained from studying rhodopsin has frequently been directly applicable to other G protein-coupled receptors we wished to ascertain if dimeric and/or oligomeric forms of the alpha(1b)-adrenoceptor could be observed and if so whether rhodopsin might provide insights into the quaternary structure of this receptor. Co-immunoprecipitation and both conventional and time-resolved fluorescence resonance energy transfer studies demonstrated quaternary structure of the alpha(1b)-adrenoceptor and, in concert with the reconstitution of fragments of this receptor, provided information on the molecular basis of these interactions. Development of three color fluorescence resonance energy transfer (FRET) allowed the imaging of alpha(1b)-adrenoceptor oligomers in single living cells. Mutation of hydrophobic residues in transmembrane domains I and IV of the receptor resulted in marked reduction in three color FRET suggesting an alteration in oligomeric organization and potential similarities with rhodopsin. The mutated alpha(1b)-adrenoceptor was unable to reach the cell surface, did not become terminally N-glycosylated and was unable to signal.

Animals↗
PubMed2002

Structural aspects of oligomerization taking place between the transmembrane alpha-helices of bitopic membrane proteins.

Recent advances in biophysical methods have been able to shed more light on the structures of helical bundles formed by the transmembrane segments of bitopic membrane proteins. In this manuscript, I attempt to review the biological importance and diversity of these interactions, the energetics of bundle formation, motifs capable of inducing oligomerization and methods capable of detecting, solving and predicting the structures of these oligomeric bundles. Finally, the structures of the best characterized instances of transmembrane alpha-helical bundles formed by bitopic membrane proteins are described in detail.

Calcium-Binding Proteins↗
PubMed2003

Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells.

In order to identify Nm23-H1's structural motifs influencing its metastasis-inhibitory activity, we transfected DU 145 human prostate carcinoma cells with the expression vector encoding the Nm23-H1 protein with mutations at the following amino acids: serine-44, a phosphorylation site; proline-96, a site corresponding to the k-pn mutation that causes developmental defects in Drosophila; and serine-120, a site of mutation in human neuroblastoma and phosphorylation. Significant decrease in colonization in soft agar and invasiveness of DU 145 cells was observed in the wild type nm23-H1 transfectants, and also in the serine-44 and serine-120 to alanine mutant nm23-H1-transfected cell lines. However, the k-pn type proline-96 to serine (P96S) and neuroblastoma type serine-120 to glycine (S120G) mutations of Nm23-H1 abrogated its inhibitory activity on colonization and invasion. Meanwhile, all of the recombinant mutant Nm23-H1 proteins produced in Escherichia coli exhibited NDP kinase activity levels at the wild type protein, although the P96S and S120G mutant proteins exhibited decreased histidine protein kinase activity and autophosphorylation level, respectively. Interestingly, only two of the mutant recombinant Nm23-H1 proteins examined, P96S and S120G, exhibited reduced hexameric and increased dimeric oligomerization relative to the wild type. These correlative data suggest that the metastasis-suppressing activity of Nm23-H1 may depend on its oligomeric structure, but not on its NDP kinase activity.

Animals↗
PubMed1990

Oligomerization and conformation change in solutions of calf lens gamma II-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles.

From analyses of the magnetic field dependence of 1/T1 (nuclear magnetic relaxation dispersion [NMRD] profiles) of water protons in solutions of highly purified calf lens gamma II-crystallin, we find that monomers form oligomers at relatively low concentrations, which increase in size with increasing concentration and decreasing temperature. At approximately 16% by volume and -4 degrees C, the mean oligomeric molecular weight is approximately 120-fold greater than the monomeric value of 20 kD. Below this concentration, there is no indication of any substantive change in conformation of the monomeric subunits. At higher concentrations, the tertiary structure of the monomer appears to reconfigure rather abruptly, but reversibly, as evidenced by the appearance of spectra-like 14N peaks in the NMRD profiles. The magnitudes of these peaks, known to arise from cross-relaxation of water protons through access to amide (NH) moieties of the protein backbone, indicate that the high concentration conformation is not compact, but open and extended in a manner that allows enhanced interaction with solvent. The data are analogous to those found for homogenates of calf and chicken lens (Beaulieu, C. F., J. I. Clark, R. D. Brown III, M. Spiller, and S. H. Koenig. 1988. Magn. Reson. Med. 8:47-57; Beaulieu, C. F., R. D. Brown III, J. I. Clark, M. Spiller, and S. H. Koenig. 1989. Magn. Reson. Med. 10:62-72). This unusually large dependence of oligomeric size and conformation on concentration in the physiological range is suggested as the mechanism by which osmotic equilibrium is maintained, at minimal metabolic expense, in the presence of large gradients of protein concentration in the lens in vivo (cf Vérétout and Tardieu, 1989. Eur. Biophys. J. 17:61-68). Finally, the results of the NMRD data provide a ready explanation of the low temperature phase transition, and "cold-cataract" separation of phases, observed in gamma II-crystallin solutions; we suggest that the phases that separate are the two major conformers detected by NMRD.

Animals↗
PubMed2001

Interdomain interactions in oligomeric enzymes: creation of asymmetry in homo-oligomers and role in metabolite channeling between active centers of hetero-oligomers.

Interdomain interactions play an important role in the structural organization of many enzymes and the conformational flexibility of their molecules. In this review, the role of intrasubunit and intersubunit domain-domain interactions in the origins of pre-existent asymmetry of homo-oligomeric D-glyceraldehyde-3-phosphate dehydrogenase and tryptophanyl-tRNA synthetase is discussed on the basis of recent X-ray data and other available information about the properties of these and related enzymes. In addition, a novel key function of interdomain interactions is considered: their potential contribution to intramolecular channeling of intermediates between active centers located on different subunits of a hetero-oligomeric enzyme (alpha,beta-heterodimeric carbamoyl phosphate synthetase).

Animals↗
PubMed2001

Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins.

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12-16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.

Animals↗
PubMed2002

The determinants of the oligomeric structure in Hsp16.5 are encoded in the alpha-crystallin domain.

The determinants of the oligomeric assembly of Hsp16.5, a small heat-shock protein (sHSP) from Methanococcus jannaschii, were explored via site-directed truncation and site-directed spin labeling. For this purpose, subunit contacts around the two-, three- and four-fold symmetry axes were fingerprinted using patterns of proximities between nitroxide spin labels introduced at selected sites. The lack of change in this fingerprint in an N-terminal truncation of the protein demonstrates that the interactions are encoded in the alpha-crystallin domain. In contrast, the truncation of the N-terminal domain of Mycobacterium tuberculosis Hsp16.3, a bacterial sHSP with an equally short N-terminal region, results in the dissociation of the oligomer to a trimer. These results, in conjunction with those from previous truncation studies in mammalian sHSP, suggest that as the alpha-crystallin domain evolved to encode a smaller basic unit than the overall oligomer, the control of the assembly and dynamics of the oligomeric structure became encoded in the N-terminal domain.

Archaeal Proteins↗
PubMed2002

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells.

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.

Adenosine↗