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PubMed2005

Homo-oligomerization of Marburgvirus VP35 is essential for its function in replication and transcription.

The nucleocapsid protein VP35 of Marburgvirus, a filovirus, acts as the cofactor of the viral polymerase and plays an essential role in transcription and replication of the viral RNA. VP35 forms complexes with the genome encapsidating protein NP and with the RNA-dependent RNA polymerase L. In addition, a trimeric complex had been detected in which VP35 bridges L and the nucleoprotein NP. It has been presumed that the trimeric complex represents the active polymerase bound to the nucleocapsid. Here we present evidence that a predicted coiled-coil domain between amino acids 70 and 120 of VP35 is essential and sufficient to mediate homo-oligomerization of the protein. Substitution of leucine residues 90 and 104 abolished (i) the probability to form coiled coils, (ii) homo-oligomerization, and (iii) the function of VP35 in viral RNA synthesis. Further, it was found that homo-oligomerization-negative mutants of VP35 could not bind to L. Thus, it is presumed that homo-oligomerization-negative mutants of VP35 are unable to recruit the polymerase to the NP/RNA template. In contrast, inability to homo-oligomerize did not abolish the recruitment of VP35 into inclusion bodies, which contain nucleocapsid-like structures formed by NP. Finally, transcriptionally inactive mutants of VP35 containing the functional homo-oligomerization domain displayed a dominant-negative phenotype. Inhibition of VP35 oligomerization might therefore represent a suitable target for antiviral intervention.

Amino Acid Sequence↗
PubMed1996

Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster.

The doublesex gene of Drosophila melanogaster encodes the alternatively spliced, sex-specific transcription factors DSXM and DSXF. These factors regulate male- and female-specific transcription of many genes. For example, female-specific transcription of the yolk protein 1 gene is regulated by DSXM repression in males and DSXF activation in females. In this study we used in vitro interaction assays and the in vivo yeast two-hybrid method to identify and examine oligomerization domains of the DSX proteins. A 66-amino-acid segment common to both proteins (amino acids 39 to 104) contains a sequence-specific DNA binding domain and an oligomerization domain (OD1). The OD1 domain oligomerizes up to at least a pentamer, but only dimers bound to a palindromic regulatory site in the yolk protein 1 gene are detected. Both subunits of the OD1 dimer are in contact with DNA. Another segment of each protein (amino acids 350 to 412 for DSXF and 350 to 427 for DSXM) contains a second oligomerization domain (OD2F and OD2M, respectively). The OD2 domains have both sex-specific and non-sex-specific sequences which are necessary for oligomerization. On the basis of sequence analysis, we predict that OD2 oligomerizes through coiled-coil interactions. We speculate that the common function of OD1 and OD2 is to oligomerize the full-length proteins, whereas their specialized functions are to form a dimeric DNA binding unit and a sex-specific transcriptional activation or repression unit.

Amino Acid Sequence↗
PubMed1999

Disruption of functions of wild-type p53 by hetero-oligomerization.

We report that a p53 segment (p53 del 1-293) containing the oligomerization domain interferes with the functions of wild-type p53. Wild-type p53 inhibits transcription mediated by human cytomegalovirus (CMV) immediate-early promoter significantly; however, co-expression of p53 del 1-293 drastically reduces this repression. We show that wild-type p53 forms hetero-oligomers with p53 del 1-293 suggesting that the hetero-oligomers are defective in repressing the CMV promoter. A synthetic promoter with p53-binding sites is transactivated significantly by wild-type p53. However, co-expression of p53 del 1-293 drastically reduces this activation. At a high concentration, a deletion mutant of wild-type p53 (del 393-327) defective in oligomerization transactivates efficiently a promoter with synthetic p53-binding sites. This transactivation remains unaffected by co-expression of p53 del 1-293. p53 del 393-327 also fails to hetero-oligomerize with p53 del 1-293 indicating that hetero-oligomerization is necessary for disruption of wild-type p53-mediated transactivation. Immunostaining experiments show that hetero-oligomerization does not lead to changes in localization of nuclear p53 demonstrating that delocalization of p53 is not the reason for inactivation. We also show that co-expression of p53 del 1-293 significantly reduces the G1/S arrest by wild-type p53 suggesting that a proper oligomeric form is necessary for wild-type p53-mediated cell cycle arrest. Thus, our work shows that hetero-oligomerization disrupts wild-type p53's biological functions and suggests a mechanism by which p53 mutants may disrupt functions of wild-type p53.

Binding Sites↗
PubMedDate not supplied

[The effect of nucleotides on oligomeric state of human lactoferrin].

A polyfunctional protein lactoferrin (LF) which is present in human barrier fluids, blood and milk and this protein of acute phase is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. Using the methods of small-angle X-ray scattering and light-scattering it was shown that LF in solution exists in oligomeric state. The level of LF oligomerization depends upon its concentration and time of keeping of no frozen neutral protein solutions. At the concentrations comparable with those in human milk (1-6 mg/ml) the average inertial radius values (Rg) of LF can reach 100-450 angstroms, while Rg for monomer LF form is 26.7 angstroms. LF was shown to complex with different nucleotides and hydrolyze them. The addition of ATP and AMP to LF demonstrating any level of oligomerization leads to increase of oligomerization processes and enhancement of the Rg values up to 600-700 angstroms According to different models of LF monomer association to its oligomeric forms (sphere, plate, cylinder) the oligomeric complexes demonstrate high Rg values which can contain from several tens up to several thousands of LF monomers. A possible role of LF oligomerization for different biological functions of the protein is discussed.

Adenosine Monophosphate↗
PubMed1990

Covalent oligomerization of rat gastric mucin occurs in the rough endoplasmic reticulum, is N-glycosylation-dependent, and precedes initial O-glycosylation.

Rat gastric mucin undergoes extensive modifications during biosynthesis, including oligomerization, N- and O-glycosylation, and sulfation. We characterized the events in the rough endoplasmic reticulum (RER) and Golgi complex and studied how these steps are interrelated, using specific inhibitors of cellular processes. The mucin precursors oligomerize in the RER by forming intermolecular disulfide bonds. The oligomers comprise a mixture of predominantly di- and trimers of molar ratio 3:2. The oligomerized precursors are transported to the Golgi complex to form mature, oligomeric mucin by extensive O-glycosylation, and sulfation. N-Glycosylation of the precursor is required for efficient oligomerization. Brefeldin A, which inhibits protein transport between RER and Golgi complex, allows oligomerization and concomitantly induces initial O-glycosylation. Oligomerization and egrees from the RER precedes initial O-glycosylation and are therefore independent of the latter process.

Animals↗
PubMed1999

Oligomerization of the EK18 mutant of the trp repressor of Escherichia coli as observed by NMR spectroscopy.

The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, l-tryptophan (Trp), at their intracellular concentrations. The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions. TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA. We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR. For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR. The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer. This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression.

Amino Acid Substitution↗
PubMed1999

Oligomerization of the influenza virus nucleoprotein: identification of positive and negative sequence elements.

The RNA genome of influenza virus is encapsidated by the virus nucleoprotein (NP) to form ribonucleoprotein (RNP) structures of defined morphology. These structures result from the ability of NP to oligomerise and to bind single-strand RNA. To characterise NP oligomerization, we developed a binding assay using immobilised NP fusion proteins and in vitro translated NP. This system was used to estimate a dissociation constant for NP-NP contacts of 2 x 10 (-7)M. Analysis of NP deletion mutants identified three sequence elements important for oligomerization. Two regions corresponding to the middle and C-terminal thirds of the polypeptide were identified as the minimal sequences capable of promoting NP-NP contacts. However, the C-terminal 23 amino-acids of NP inhibited oligomerization, as their removal increased self-association 10-fold. Single codon changes identified amino acids important for the function of these regions. Alanine substitution of R199 decreased binding affinity threefold, whereas alteration of R416 had a more drastic effect, reducing binding >10-fold. In contrast, mutation of F479 increased self-association fivefold. Mutations altering NP oligomerization affected the ability of the polypeptides to support influenza virus gene expression in an in vivo assay. Decreased oligomerization activity correlated with decreased transcriptional function. However, mutations that increased self-association also decreased transcription competence. This indicates that NP contains both positive and negative sequence elements involved in oligomerization and is consistent with the importance of NP-NP contacts for the formation of a transcriptionally active RNP.

Amino Acid Substitution↗
PubMed1989

Stability of human interferon-beta 1: oligomeric human interferon-beta 1 is inactive but is reactivated by monomerization.

Human interferon-beta 1 is extremely stable is a low ionic strength solution of pH 2 such as 10 mM HCl at 37 degrees C. However, the presence of 0.15 M NaCl led to a remarkable loss of antiviral activity. The molecular-sieve high-performance liquid chromatography revealed that, whereas completely active human interferon-beta 1 eluted as a 25 kDa species (monomeric form), the inactivated preparation eluted primarily as a 90 kDa species (oligomeric form). The specific activity (units per mg protein) of the oligomeric form was approx. 10% of that of the monomeric form. This observation shows that oligomeric human interferon-beta 1 is apparently in an inactive form. When the oligomeric eluate was resolved by polyacrylamide gel containing sodium dodecyl sulphate (SDS), it appeared to be monomeric under non-reducing conditions. Monomerization of the oligomeric human interferon-beta 1 by treatment with 1% SDS, fully regenerated its antiviral activity. These results suggest that the inactivation of the human interferon-beta 1 preparation was caused by its oligomerization via hydrophobic interactions without the formation of intermolecular disulphide bonds. These oligomers can be dissociated by SDS to restore biological activity.

Chromatography, High Pressure Liquid↗
PubMed2005

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.

Affinity Labels↗
PubMed2005

Adenosine A2a receptor/dopamine D2 receptor hetero-oligomerization: a hypothesis that may explain behavioral sensitization to psychostimulants and schizophrenia.

The mechanisms underlying psychostimulant-induced behavioral sensitization and schizophrenia are yet to be fully elucidated. Evidence suggests that the dopamine D2 receptor (DRD2) as well as other neurotransmitter system receptors may be involved in the two conditions, and previous reports have hypothesized that oligomerization of dopamine receptors and analogs from other neurotransmitter systems may underlie the mechanism responsible. This paper describes findings which show co-localization of DRD2 and the adenosine A2a receptor (A2aAR) in the striatum, interaction between the two receptors and A2aAR/DRD2 hetero-oligomerization in the neuronal cell. The possibility that A2aAR/DRD2 hetero-oligomerization may be involved in the development of psychostimulant-induced behavioral sensitization or the pathogenesis of schizophrenia is explored. A2aAR/DRD2 hetero-oligomerization can enhance dopaminergic function, resulting in an increased behavioral response to psychostimulants or the development of psychotic symptoms. Receptor oligomerization can affect receptor degradation, which may affect the persistence of behavioral sensitization or psychotic symptoms. The proposed A2aAR/DRD2 hetero-oligomerization model may suggest new directions in the understanding and treatment of schizophrenia.

Animals↗
PubMed2005

Factors affecting oligomerization status of UDP-glucose pyrophosphorylase.

UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6-9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.

Buffers↗
PubMed2005

Protein kinase C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50.

Ezrin-Radixin-Moesin (ERM) binding phosphoprotein 50 (EBP50, a.k.a. NHERF-1) is a scaffold protein essential for the localization and coordinated activity of apical transporters, enzymes and receptors in epithelial cells. EBP50 acts via multiple protein binding interactions, including oligomerization through interactions of its PSD95-Dlg-ZO1 (PDZ) domains. EBP50 can be phosphorylated on multiple sites and phosphorylation of specific sites modulates the extent of oligomerization. The aim of the present study was to test the capacity of protein kinase C (PKC) to phosphorylate EBP50 and to regulate its oligomerization. In vitro experiments showed that the catalytic subunit of PKC directly phosphorylates EBP50. In HEK-293 cells transfected with rat EBP50 cDNA, a treatment with 12 myristate 13-acetate (PMA) induced a translocation of PKCalpha and beta isoforms to the membrane and increased 32P incorporation into EBP50. In co-transfection/co-precipitation studies, PMA treatment stimulated EBP50 oligomerization. Mass spectrometry analysis of full-length EBP50 and phosphorylation analyses of specific domains, and of mutated or truncated forms of EBP50, indicated that PKC-induced phosphorylation of EBP50 occurred on the Ser337/Ser338 residue within the carboxyl-tail domain of the protein. Truncation of Ser337/Ser338 also diminished PKC-induced oligomerization of EBP50. These results suggest the PKC signaling pathway can impact EBP50-dependent cellular functions by regulating EBP50 oligomerization.

Amino Acid Sequence↗
PubMed1999

A fluorescence energy transfer method for analyzing protein oligomeric structure: application to phospholamban.

We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, was used to determine the dependence of FET on the number of subunits, the distance between labeled sites on different subunits, and the fraction of subunits remaining monomeric. By measuring FET as a function of the donor/acceptor ratio, the above parameters of the oligomeric structure can be resolved over a substantial range of their values. We used this approach to investigate the oligomeric structure of phospholamban (PLB), a 52-amino acid protein in cardiac sarcoplasmic reticulum (SR). Phosphorylation of PLB regulates the SR Ca-ATPase. Because PLB exists primarily as a homopentamer on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, this hypothesis must be tested by determining directly the oligomeric structure of PLB in the lipid membrane. To accomplish this goal, PLB was labeled at Lys-3 in the cytoplasmic domain, with two different amine-reactive donor/acceptor pairs, which gave very similar FET results. In detergent solutions, FET was not observed unless the sample was first boiled to facilitate subunit mixing. In lipid bilayers, FET was observed at 25 degrees C without boiling, indicating a dynamic equilibrium among PLB subunits in the membrane. Analysis of the FET data indicated that the dye-labeled PLB is predominantly in oligomers having at least 8 subunits, that 7-23% of the PLB subunits are monomeric, and that the distance between dyes on adjacent PLB subunits is about 10 A. A point mutation of PLB (L37A) that runs as monomer on SDS-PAGE showed no energy transfer, confirming its monomeric state in the membrane. We conclude that FET is a powerful approach for analyzing the oligomeric structure of PLB, and this method is applicable to other oligomeric proteins.

Animals↗
PubMed2002

Mechanism of oligomerization of Escherichia coli carbamoyl phosphate synthetase and modulation by the allosteric effectors. A site-directed mutagenesis study.

We use site-directed mutagenesis to clarify the role of effector-mediated oligomerization changes on the modulation of the activity of Escherichia coli carbamoyl phosphate synthetase (CPS) by its allosteric activator ornithine and its inhibitor UMP. The regulatory domain mutations H975L, L990A and N992A abolished, and N987V decreased CPS oligomerization. The oligomerization domain mutation L421E prevented tetramer but not dimer formation. None of the mutations had drastic effects on enzyme activity or changed the sensitivity or apparent affinity of CPS for ornithine and UMP. Our findings exclude the involvement of oligomerization changes in the control of CPS activity, and show that CPS dimers are formed by the interactions across regulatory domains, and tetramers by the interactions of two dimers across the oligomerization domains. A mechanism for effector-mediated changes of the oligomerization state is proposed.

Allosteric Regulation↗
PubMed2000

Oligomerization of mouse alpha 1-syntrophin and self-association of its pleckstrin homology domain 1 containing sequences.

Syntrophins are known to self-associate to form oligomers. Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and were found to also oligomerize and in a micromolar Ca(2+)-dependent manner. The oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to self-associate, and calmodulin or Ca(2+)-chelating agents such as ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Since calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca(2+), Ca(2+) binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.

Animals↗
PubMed1985

Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor.

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.

Animals↗
PubMed2002

Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins.

2-Cys peroxiredoxins (Prxs) are a large and diverse family of peroxidases which, in addition to their antioxidant functions, regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers (alpha(2)), utilizing a unique intermolecular redox-active disulfide center for the reduction of peroxides, and are known to form two oligomeric states: individual alpha(2) dimers or doughnut-shaped (alpha(2))(5) decamers. Here we characterize both the oligomerization properties and crystal structure of a bacterial 2-Cys Prx, Salmonella typhimurium AhpC. Analytical ultracentrifugation and dynamic light scattering show that AhpC's oligomeric state is redox linked, with oxidization favoring the dimeric state. The 2.5 A resolution crystal structure (R = 18.5%, R(free) = 23.9%) of oxidized, decameric AhpC reveals a metastable oligomerization intermediate, allowing us to identify a loop that adopts distinct conformations associated with decameric and dimeric states, with disulfide bond formation favoring the latter. This molecular switch contains the peroxidatic cysteine and acts to buttress the oligomerization interface in the reduced, decameric enzyme. A structurally detailed catalytic cycle incorporating these ideas and linking activity to oligomeric state is presented. Finally, on the basis of sequence comparisons, we suggest that the enzymatic and signaling activities of all 2-Cys Prxs are regulated by a redox-sensitive dimer to decamer transition.

Amino Acid Sequence↗
PubMed2004

The region adjacent to the highly immunogenic site and shielded by the middle domain is responsible for self-oligomerization/client binding of the HSP90 molecular chaperone.

We here investigated the mechanism of self-oligomerization of the 90-kDa heat shock protein (HSP90) molecular chaperone, because it is known that this oligomerization reflects the client-binding activity. The transition temperatures for the self-oligomerization of the full-length forms of human HSP90alpha and HtpG (bacterial HSP90), i.e., 45 and 60 degrees C, respectively, were identical to those for the dissociation of the recombinant N domain (residues 1-400 of human HSP90alpha and residues 1-336 of HtpG in our definition) from the remainder of the molecule. The N domain of human HSP90alpha expressed in Escherichia coli was oligomeric, and the oligomerization activity was localized within residues 311-350, i.e., C-terminally adjacent to the highly immunogenic site (residues 291-304). Particularly, residues 341-350 were critical on oligomerization. On the other hand, residues 289-389 were indispensable for the interaction with the M domain (residues 401-618) of the molecule. Oligomer formation of the N domain was efficiently suppressed by its extension until Lys546, i.e., residues 401-546, which is required for the interaction with the N domain. Among highly conserved amino acids at residues 289-400, Trp297, Pro379, and Phe384 were essential for the interaction with the M domain. With these observations taken together, we propose as the activation mechanism of HSP90 molecular chaperone that heat stress induces the liberation of the oligomerization/client-binding site of residues 311-350 by disrupting the intramolecular interaction between residues 289-389 and 401-546.

Bacterial Proteins↗