Crystal structure of the tetrameric inositol 1-phosphate phosphatase (TM1415) from the hyperthermophile, Thermotoga maritima

The structure of the first tetrameric inositol monophosphatase (IMPase) has been solved. This enzyme, from the eubacterium Thermotoga maritima, similarly to its archaeal homologs exhibits dual specificity with both IMPase and fructose-1,6-bisphosphatase activities. The tetrameric structure of this unregulated enzyme is similar, in its quaternary assembly, to the allosterically regulated tetramer of fructose-1,6-bisphosphatase. The individual dimers are similar to the human IMPase. Additionally, the structures of two crystal forms of IMPase show significant differences. In the first crystal form, the tetrameric structure is symmetrical, with the active site loop in each subunit folded into a beta-hairpin conformation. The second form is asymmetrical and shows an unusual structural change. Two of the subunits have the active site loop folded into a beta-hairpin structure, whereas in the remaining two subunits the same loop adopts an alpha-helical conformation.     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Construction and analyses of tetrameric forms of yeast NAD+-specific isocitrate dehydrogenase

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation with an IDH1 G15D or IDH1 D168K residue substitution produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ～50% reductions in V(max) and in cooperativity with respect to isocitrate relative to those of the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated (-5)IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit of one tetramer to the IDH2 Cys-150 residues in the other tetramer.     

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at 相似文献   

Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions.

The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.     

A pH-dependent aquomet-to-hemichrome transition in crystalline horse methemoglobin

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.     

Unfolding thermodynamics of the tetrameric chaperone, SecB

SecB is a cytosolic tetrameric chaperone in Escherichia coli, which maintains polypeptides, destined for export in a translocation competent state. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. Increasing the pH decreases the stability of the tetramer significantly, the T(m) changing from 341.3 K at pH 6.5 to 332.6 K at pH 9.5. The value of DeltaC(p) obtained from measurements of DeltaH(m) as a function of T(m) was 10.7 +/- 0.7 kcal mol(-1) K(-1). The value of DeltaC(p) is among the highest measured for a multimeric protein. At 298 K, pH 7.4, the DeltaG degrees (u) for the SecB tetramer is 27.9 +/- 2 kcal mol(-1). Denaturant-mediated unfolding of SecB was found to be irreversible. The reactivity of the four solvent-exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well-folded, and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity.     

Functional assembly of AMPA and kainate receptors is mediated by several discrete protein-protein interactions

Functional heterogeneity of ionotropic glutamate receptors arises not only from the existence of many subunits and isoforms, but also from combinatorial assembly creating channels with distinct properties. This heteromerization is subtype restricted and thought to be determined exclusively by the proximal extracellular N-terminal domain of the subunits. However, using functional assays for heteromer formation, we show that, besides the N-terminal domain, the membrane sector and the C-terminal part of S2 are critical determinants for the formation of functional channels. Our results are compatible with a model where the N-terminal domain only mediates the initial subunit associations into dimers, whereas for the assembly of the full functional tetramer, compatibility of the other regions is required.     

Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM

F factor TraM is essential for efficient bacterial conjugation, but its molecular function is not clear. Because the physical properties of TraM may provide clues to its role in conjugation, we have characterized the TraM oligomerization equilibrium. We show that the reversible unfolding transition is non-two-state, indicating the presence of at least one intermediate. Analytical ultracentrifugation experiments indicate that the first phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state in the second phase. Furthermore, we show that a C-terminal domain isolated by limited proteolysis is tetrameric in solution, like the full-length protein, and that its loss of structure correlates with dissociation of the TraM tetramer. Unfolding of the individual domains indicates that the N- and C-terminal regions act cooperatively to stabilize the full-length protein. Together, these experiments suggest structural overlap of regions important for oligomerization and DNA binding. We propose that modulating the oligomerization equilibrium of TraM may regulate its essential activity in bacterial conjugation.     

Substitution of the heme binding module in hemoglobin alpha- and beta-subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.     

Immobilised monomers of human liver arginase.

Human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was immobilised by attachment to nylon with glutaraldehyde as a crosslinking agent. Incubation of the immobilised tetrameric enzyme with EDTA followed by dialysis resulted in the dissociation of the enzyme into inactive matrix-bound and solubilised subunits. Both species recovered enzymatic activity after incubation with Mn2+, and the activity of the reactivated matrix-bound subunits was nearly 25% of that shown by the enzyme initially attached to the support in the tetrameric form. When the reactivated bound subunits were incubated with soluble subunits in the presence of Mn2+, they 'picked-up' from the solution an amount of protein and enzymatic activity almost identical to that initially lost by the immobilised tetramer after the dissociating treatment with EDTA. This occurred only in the presence of Mn2+. It is suggested that the reactivation of the subunits of arginase involves the initial formation of an active monomer, which then acquires a conformation that favours a reassociation to the tetrameric state.     

Folding of Tetrameric p53: Oligomerization and Tumorigenic Mutations Induce Misfolding and Loss of Function

The physiologically active form of p53 consists of a tetramer of four identical 393-amino-acid subunits associated via their tetramerization domains (TDs; residues 325-355). One in two human tumors contains a point mutation in the DNA binding domain (DBD) of p53 (residues 94-312). Most existing studies on the effects of these mutations on p53 structure and function have been carried out on the isolated DBD fragment, which is monomeric. Recent structural evidence, however, suggests that DBDs may interact with each other in full-length tetrameric forms of p53. Here, we investigate the effects of tumorigenic DBD mutations on the folding of p53 in its tetrameric form. We employ the construct consisting of DBD and TD (amino acids 94-360). We characterize the stability and conformational state of the tumorigenic DBD mutants R248Q, R249S, and R282Q using equilibrium denaturation and functional assays. Destabilizing mutations cause DBD to misfold when it is part of the p53 tetramer, but not when it is monomeric. This conformation is populated under moderately destabilizing conditions (10 °C in 2 M urea, and at physiological temperature in the absence of denaturant). Under those same conditions, it is not present in the isolated DBD fragment or in the presence of the TD mutation L344P, which abolishes tetramerization. Misfolding appears to involve intramolecular DBD-DBD association within a single tetrameric molecule. This association is promoted by destabilization of DBD (caused by mutation or elevated temperature) and by the high local DBD concentration enforced by tetramerization of TD. Disrupting the nonnative DBD-DBD interaction or transiently inhibiting tetramerization and allowing p53 to fold as a monomer may be potential strategies for pharmacological intervention in cancer.     

Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P_i to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.     

Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively

The structures of D-xylose isomerase from Arthrobacter strain B3728 containing the polyol inhibitors xylitol and D-sorbitol have been solved at 2.5 A and 2.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the D-sorbitol (xylitol) bound molecules, for 43,615 (32,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a dimer, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu216, Asp244 and Asp292. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-2 and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a dimer of "active" dimers, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp23 and Arg139, appears to play a key role in stabilizing the active dimer and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of α and β subunits and forms a “jellyfish-like” structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two α and two β subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of α (α1 and α2) and β subunits (β1 and β2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD β1 subunit at 1.9 Å resolution and its functional analysis. TsPFD β1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The β hairpin linkers of β1 subunits assemble to form a β barrel “body” around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the β1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric β1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD β1 subunits act as molecular chaperones in living cells of some archaea.     

Crystal structure of sorbitol dehydrogenase

Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20%. SDH is a tetramer with one zinc ion per subunit. We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object. The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH. The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii. The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site. The SDH molecule can be considered to be a dimer of dimers, with subunits A-B and C-D, where the dimer interactions are similar to those in mammalian ADH. The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side. In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.     

Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition

Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R- to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R- to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.     

X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.