Stable octameric structure of recombinant hemoglobin alpha(2)beta(2)83 Gly-->Cys

We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.     

Construction of molecular assemblies via docking: modeling of tetramers with D2 symmetry

Comparative modeling methods are commonly used to construct models of homologous proteins or oligomers. However, comparative modeling may be inapplicable when the number of subunits in a modeled oligomer is different than in the modeling template. Thus, a dimer cannot be a template for a tetramer because a new monomer-monomer interface must be predicted. We present in this study a new prediction approach, which combines protein-protein docking with either of two tetramer-forming algorithms designed to predict the structures of tetramers with D2 symmetry. Both algorithms impose symmetry constraints. However, one of them requires identification of two of the C2 dimers within the tetramer in the docking step, whereas the other, less demanding algorithm, requires identification of only one such dimer. Starting from the structure of one subunit, the procedures successfully reconstructed 16 known D2 tetramers, which crystallize with either a monomer, a dimer or a tetramer in the asymmetric unit. In some cases we obtained clusters of native-like tetramers that differ in the relative rotation of the two identical dimers within the tetramer. The predicted structural pliability for concanavalin-A, phosphofructokinase, and fructose-1,6-bisphosphatase agrees semiquantitatively with the observed differences between the several experimental structures of these tetramers. Hence, our procedure identifies a structural soft-mode that allows regulation via relative rigid-body movements of the dimers within these tetramers. The algorithm also predicted three nearly correct tetramers from model structures of single subunits, which were constructed by comparative modeling from subunits of homologous tetrameric, dimeric, or hexameric systems.     

The three-dimensional structure of diaminopimelate decarboxylase from Mycobacterium tuberculosis reveals a tetrameric enzyme organisation

The three-dimensional structure of the enzyme diaminopimelate decarboxylase from Mycobacterium tuberculosis has been determined in a new crystal form and refined to a resolution of 2.33 Å. The monoclinic crystals contain one tetramer exhibiting D₂-symmetry in the asymmetric unit. The tetramer exhibits a donut-like structure with a hollow interior. All four active sites are accessible only from the interior of the tetrameric assembly. Small-angle X-ray scattering indicates that in solution the predominant oligomeric species of the protein is a dimer, but also that higher oligomers exist at higher protein concentrations. The observed scattering data are best explained by assuming a dimer–tetramer equilibrium with about 7% tetramers present in solution. Consequently, at the elevated protein concentrations in the crowded environment inside the cell the observed tetramer may constitute the biologically relevant functional unit of the enzyme.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Evolution of different oligomeric glycyl-tRNA synthetases

There are two oligomeric types of glycyl-tRNA synthetases (GlyRSs) in genome, the alpha2beta2 tetramer and alpha2 dimer. Here, we showed that the anticodon-binding domains (ABDs) of dimeric and tetrameric GlyRSs are non-homologous, although their catalytic central domains (CCDs) are homologous. The dimeric GlyRS_ABD is fused to the C-terminal of CCD in alpha-subunit, but the tetrameric GlyRS_ABD is to the C-terminal in beta-subunit during evolution. Generally, one species only contains one oligomeric type of GlyRS, but the both oligomeric GlyRSs with the multiple homologous domains can be observed in Magnetospirillum magnetotacticum genome, nevertheless, these homologous domains are probably from different genomes.     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.     

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.     

Interaction of flavin mononucleotide with dimeric and tetrameric forms of muscle phosphorylase beta.

Interaction of flavin mononucleotide (FMN) with dimeric and tetrameric forms of rabbit muscle glycogen phosphorylase beta has been studied under the conditions when allosteric activator binding sites are saturated by AMP (1 mM AMP; pH 6.8; 17 degrees C). Simultaneous use of schlieren optical system and photoelectric scanning absorption optical system of analytical ultracentrifuge Spinco, model E, makes it possible to register the oligomeric state of the enzyme and calculate the degree of saturation of individual oligomeric enzyme forms by FMN. The apparent association constant for the equilibrium dimer in equilibrium with tetramer decreased with increasing FMN concentration. The microscopic dissociation constants for the complexes of dimeric and tetrameric forms of glycogen phosphorylase beta with FMN have been found to be equal to 10 and 79 microM, respectively.     

Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI–TOF–TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.     

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer-dimer interaction and prevent the formation of the tetramer.     

Subunit disassembly and unfolding kinetics of hemoglobin studied by time-resolved electrospray mass spectrometry

We report the use of electrospray ionization (ESI) mass spectrometry (MS) in conjunction with online rapid mixing to monitor the kinetics of acid-induced ferrihemoglobin denaturation. Under equilibrium conditions, the hemoglobin mass spectrum is dominated by the intact heterotetramer. Dimeric and monomeric species are also observed at lower intensities. In addition, ionic signals corresponding to hexameric (tetramer-dimer) and octameric (tetramer x 2) hemoglobin species are observed. These complexes may represent weak solution-phase assemblies. The acid-induced denaturation process was monitored for reaction time ranging from 9 ms to approximately 3 s. The data obtained were subjected to a global analysis procedure which simultaneously fit all kinetic (ESI-MS intensity vs time) profiles to multiexponential expressions. Results of the global analysis are consistent with the coexistence of two subpopulations of tetrameric hemoglobin which differ in their disassembly rates and ESI charge states. The higher-charge state tetramer ions preferentially dissociate via a rapid pathway (tau(1) = 51 ms), resulting in the transient formation of a heme-saturated dimer, holo-alpha-globin, and a heme-deficient dimer. The latter is shown by MS/MS to be comprised of a heme-bound alpha-subunit complexed with an apo-beta-chain. The slow-decaying tetramer population, apparent at a slightly lower average charge state, breaks down into its monomeric constituents with no observable intermediate species (tau(2) = 390 ms). Surprisingly, unfolded apo-alpha-globin is formed more rapidly than unfolded apo-beta-globin. The appearance of the latter occurs with a relaxation time tau(3) of 1.2 s. It is postulated that accumulation of unfolded apo-beta-globin is delayed by transient population of an undetected unfolding intermediate.     

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Oligomerization of ribonuclease A: two novel three-dimensional domain-swapped tetramers

By lyophilization from 40% acetic acid solutions, bovine ribonuclease A forms several types of three-dimensional domain-swapped oligomers: dimers, trimers, tetramers, and higher order multimers. Each oligomeric species comprehends at least two conformers: one less basic and one more basic. The structures of the two dimers and one trimer have been solved. Plausible models have been proposed for the other oligomers. Among them, all chromatographic patterns show the constant presence of minority species, and we focused our attention on two of them. The first oligomer (named X) elutes between the two trimeric conformers; the second (named Y) elutes as a shoulder in the ascending limb of the more basic trimer. After purification with cation-exchange chromatography, on the basis of (a) gel filtration analyses, (b) gel electrophoreses under nondenaturing conditions, (c) SDS-PAGE, (d) cross-linking experiments with divinylsulfone and 1,5-difluoro 2,4-dinitrobenzene, (e) enzymatic activity assays, (f) identification of the products of their spontaneous dissociation, and (g) controlled proteolysis with subtilisin, we propose that the X and Y oligomeric species contain two novel three-dimensional domain-swapped tetrameric conformers of RNase A, differing from each other as well as from the two tetramers already identified. For the two novel tetramers we showed tentative structural models. X(TT) could be a circular NCNC-tetramer; Y(TT) could be a propeller-like C-trimer with an attached N-swapping monomer (NCCC(TT)), identical to a model proposed by Liu and Eisenberg (Liu, Y., and Eisenberg, D. (2002) Protein Sci. 11, 1285-1299).     

Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium

We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the homotetrameric species exchanged dimers, because when the mixture was analyzed both by size exclusion chromatography and native polyacrylamide gel electrophoresis a third hybrid tetrameric species was detected. Furthermore, treatment of SecB with 5,5'-dithiobis-(2-nitrobenzoic acid), which modifies the sulfhydryl group on cysteines, caused irreversible dissociation to a dimer indicating that cysteine must be involved in the stabilizing interactions at the dimer interface. It is clear that the two dimer-dimer interfaces of the SecB tetramer are differentially stable. Dissociation at one interface allows for a dynamic dimer-tetramer equilibrium. Because only dimers were exchanged it is clear that the other interface between dimers is significantly more stable, otherwise oligomers should have formed with a random distribution of monomers.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B

Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.     

Structural insights into the pre-amyloid tetramer of β-2-microglobulin from covalent labeling and mass spectrometry

The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.     

Rattlesnake hemoglobins: Functional properties and tetrameric stability

The present work analyzed the tetrameric stability of the hemoglobins from the rattlesnake C. durissus terrificus using analytical gel filtration chromatography, SAXS and osmotic stress. We show that the dissociation mechanism proposed for L. miliaris hemoglobin does not apply for these hemoglobins, which constitute stable tetramers even at low concentrations.     

Distinguishing between functional monomeric and oligomeric complexes of the Ca,Mg-ATPase in sarcoplasmic reticulum

Techniques are described for using blocking agents to distinguish between enzymes which are functional monomers and oligomers. To achieve this distinction the blocking agent must react exclusively at the active site with a stoichiometry of one mole of site per mole enzyme. The effect of the blocking agent on enzymatic activity in oligomers of n = 2 and 4 are described and the optimal degree of blocking is considered for tests of enzyme activity at saturating and less than saturating substrate concentrations. For saturating concentrations and a dimer the distinction between dimer and monomer is best observed with 50 per cent of sites blocked. For a tetramer the distinction is best made at higher degrees of blockade. The use of saturating substrate concentrations is thus limited to small oligomers. If nonsaturating substrate concentrations are used and normalized double reciprocal plots of the dependence of enzyme activity on substrate concentrations are made then the distinction between monomer and oligomer can readily be made for dimers, tetramers, and higher n-mers. The principles developed to distinguished monomeric from oligomeric enzymes are applied to published data obtained with the Ca Mg-ATPase of sarcoplasmic reticulum. Fluorescein isothiocyanate is the blocking agent. Plots of the published data support both the monomeric and tetrameric models for allosteric regulation with the preponderance of the data supporting the monomeric model.