All hierarchical levels are involved in conformational transitions of the 4 x 6-meric tarantula hemocyanin upon oxygenation

The respiratory protein of the tarantula Eurypelma californicum is a 4 x 6-meric hemocyanin that binds oxygen with high cooperativity. This requires the existence of different conformations which have been confirmed by small angle X-ray scattering (SAXS). Here we present reconstructed 3D-models of the oxy- and deoxy-forms of tarantula hemocyanins, as obtained by fitting small angle X-rays scattering curves on the basis of known X-ray structures and electron microscopy of related hemocyanins. For the first time, the involvement of movements at all levels of the quaternary structure was confirmed for an arthropod hemocyanin upon oxygenation. The two identical 2 x 6-meric half-molecules of the native 4 x 6-mer were shifted in the oxy-state along each other compared with the deoxy-state by about 14 A. In addition, the angle between the two 2 x 6-meric half-molecules increased by 13 degrees. Within these 2 x 6-mers the two hexamers were rotated against each other by about 26 degrees with respect to the deoxy-state. In addition, the distance between the two trimers of each hexamer increased upon oxygenation by about 2.5 A. These strongly coupled movements are based on the particular hierarchical structure of the 4 x 6-mer. It also shows a concept of allosteric interaction in hierarchically assembled proteins to guarantee the involvement of all subunits of a native oligomer to establish very high Hill coefficients.     

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.     

Limulus polyphemus hemocyanin: 10 A cryo-EM structure, sequence analysis, molecular modelling and rigid-body fitting reveal the interfaces between the eight hexamers

The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1x6mers) that form the native 8x6mer in a nested hierarchy of 2x6mers and 4x6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1x6mer are available, but for the 8x6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8x6mer are not firmly established, and the molecular interfaces between the eight hexamers are still to be defined. This, however, is crucial for understanding how allosteric transitions are mediated between the different levels of hierarchy. Here, we show the 10 A structure (FSC(1/2-bit) criterion) of the oxygenated 8x6mer from cryo-electron microscopy (cryo-EM) and single-particle analysis. Moreover, we show its molecular model as obtained by DNA sequencing of subunits II, IIIA, IV and VI, and molecular modelling and rigid-body fitting of all subunit types. Remarkably, the latter enabled us to improve the resolution of the cryo-EM structure from 11 A to the final 10 A. The 10 A structure allows firm assessment of various structural parameters of the 8x6mer, the 4x6mer and the 2x6mer, and reveals a total of 46 inter-hexamer bridges. These group as 11 types of interface: four at the 2x6mer level (II-II, II-IV, V-VI, IV-VI), three form the 4x6mer (V-V, V-VI, VI-IIIB/IV/V), and four are required to assemble the 8x6mer (IIIA-IIIA, IIIA-IIIB, II-IV, IV-IV). The molecular model shows the amino acid residues involved, and reveals that several of the interfaces are intriguingly histidine-rich and likely to transfer allosteric signals between the different levels of the nested hierarchy.     

Reaction pathway for the quaternary structure change in hemoglobin

We perform a computer simulation of the quaternary structure change during the allosteric transition of hemoglobin. The simulation is based on a docking procedure by which αβ dimers of human hemoglobin are associated into tetramers after being rotated in various orientations. The stability of tetramers thus reconstituted is estimated from the values of a simplified energy function describing nonbonded interactions and from the area of the surface buried in dimer–dimer contacts (their interface area), which we take to represent stabilizing interactions and solvent contribution. A systematic analysis of tetramers reconstituted with twofold symmetry reveals that when the dimers have the R tertiary structure, only tetramers having R-like quaternary structures are stable. When the dimers have the T tertiary structure, they may associate into T-like tetramers or a variety of quaternary structures ranging from T to near R, thus tracing a plausible reaction pathway for the allosteric transition. We subject intermediates of this pathway to energy refinement with rigid αβ dimers. The refinement demonstrates that symmetrical structures are more stable than non symmetrical ones. A detailed analysis of dimer–dimer contacts in intermediates shows how close packing is maintained over large interfaces throughout the quaternary structure change, especially in the “switch region” of contact between the C helix of α-chains and the FG corner of β-chains.     

Novel dissociation mechanism of a polychaetous annelid extracellular haemoglobin

The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of approximately 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or 'linker' subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS/PAGE, UV-visible spectrophotometry, electrospray-ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

Modes of attachment of acetylcholinesterase to the surface membrane

Acetylcholinesterase (AChE) occurs in multiple molecular forms differing in their quaternary structure and mode of anchoring to the surface membrane. Attachment is achieved by post-translational modification of the catalytic subunits. Two such mechanisms are described. One involves attachment to catalytic subunit tetramers, via disulfide bridges, of a collagen-like fibrous tail. This, in turn, interacts, primarily via ionic forces, with a heparin-like proteoglycan in the extracellular matrix. A second such modification involve the covalent attachment of a single phosphatidylinositol molecule at the carboxyl-terminus of each catalytic subunit polypeptide; the diacylglycerol moiety of the phospholipid serves to anchor the modified enzyme hydrophobically to the lipid bilayer of the plasma membrane. The detailed molecular structure of these two classes of acetylcholinesterase are discussed, as well as their biosynthesis and mode of anchoring.     

Mechanism of substrate inhibition in Escherichia coli phosphofructokinase

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP. In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition. Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms. We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3). The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates. In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer. Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon. The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.     

Multiple phosphate positions in the catalytic site of glycogen phosphorylase: structure of the pyridoxal-5''-pyrophosphate coenzyme-substrate analog.

The three-dimensional structure of an R-state conformer of glycogen phosphorylase containing the coenzyme-substrate analog pyridoxal-5'-diphosphate at the catalytic site (PLPP-GPb) has been refined by X-ray crystallography to a resolution of 2.87 A. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R-state PLPP-GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609-616), the tertiary structures differ in that the two domains of the PLPP-GPb subunits are rotated apart by 5 degrees relative to the T-state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R-state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R-state.     

Allosteric regulation and communication between subunits in uracil phosphoribosyltransferase from Sulfolobus solfataricus

Uracil phosphoribosyltransferase (UPRTase) catalyzes the conversion of 5-phosphate-alpha-1-diphosphate (PRPP) and uracil to uridine 5'-monophosphate (UMP) and diphosphate. The UPRTase from Sulfolobus solfataricus has a unique regulation by nucleoside triphosphates compared to UPRTases from other organisms. To understand the allosteric regulation, crystal structures were determined for S. solfataricus UPRTase in complex with UMP and with UMP and the allosteric inhibitor CTP. Also, a structure with UMP bound in half of the active sites was determined. All three complexes form tetramers but reveal differences in the subunits and their relative arrangement. In the UPRTase-UMP complex, the peptide bond between a conserved arginine residue (Arg80) and the preceding residue (Leu79) adopts a cis conformation in half of the subunits and a trans conformation in the other half and the tetramer comprises two cis-trans dimers. In contrast, four identical subunits compose the UPRTase-UMP-CTP tetramer. CTP binding affects the conformation of Arg80, and the Arg80 conformation in the UPRTase-UMP-CTP complex leaves no room for binding of the substrate PRPP. The different conformations of Arg80 coupled to rearrangements in the quaternary structure imply that this residue plays a major role in regulation of the enzyme and in communication between subunits. The ribose ring of UMP adopts alternative conformations in the cis and trans subunits of the UPRTase-UMP tetramer with associated differences in the interactions of the catalytically important Asp209. The active-site differences have been related to proposed kinetic models and provide an explanation for the regulatory significance of the C-terminal Gly216.     

Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.     

Immunological correspondence between arthropod hemocyanin subunits. II. Xiphosuran (Limulus) and spider (Eurypelma, Cupiennius) hemocyanin

The hemocyanins of the horseshoe crab Limulus polyphemus (48-mer), the tarantula Eurypelma californicum (24-mer), and the lycosid spider Cupiennius salei (dodecamer, hexamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Among the subunits a to g of Eurypelma on the one side, and I to VI of Limulus on the other, a number of cross-reactions were obtained which agree with the topologic subunit positions in the published models of quaternary structure: a = II, b-c = V-VI, d = IV, e = I, f = IIIb, g = IIIa (IIa). However, cross-reactivity was only strong in the following combinations: a/II, d/IV, b-c/V-VI (the monomers of the two heterodimers could not be correlated individually). A rather weak cross-reaction was obtained in the case of e/I and g/IIIa (IIa); a cross-reaction between f and IIIb was almost undetectable. On the other hand, f/IV clearly cross-reacted, and so did e/IIIa (IIa), which apparently is not in agreement with the two models of quaternary structure. These unexpected relationships, however, indicate the possible phylogeny of the subunits. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunit IV of Limulus and, moreover, revealed common antigen determinants present on these subunits. Denaturation of hemocyanin subunits of the three species with 8M urea yielded a completely different immunological behavior in that in all intra- and interspecific combinations the reaction of immunological identity was obtained. The published models of quaternary structure and a possible subunit phylogeny of cheliceratan hemocyanins is discussed in view of the present results and the results of the preceding paper. [Markl, J. et al. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 619-631.]     

Quaternary structure of pig liver phosphofructokinase

The quaternary structure of phosphofructokinase from pig liver has been studied by electron microscopy. Particles ranging in size from tetramers to long flexible chains of tetramers were commonly observed. Phosphofructokinase tetramers are square planar and approximately 110 A on a side; individual subunits are roughly spherical, with a mean radius of 28 A. Chains are formed by end-to-end association of tetramers rather than by tetramer stacking. The geometry of association implies that phosphofructokinase tetramers possess D2 symmetry, with distinct isologous bonding domains for dimer, tetramer, and chain formation.     

Nested allostery of arthropodan hemocyanin (Eurypelma californicum and Homarus americanus). The role of protons

Continuous oxygen binding curves for two arthropodan hemocyanins were performed at different pH values ranging from 7.0 to 8.7 and in the presence of physiological concentrations of the bivalent ions Ca2+ and Mg2+. The arthropods Eurypelma californicum and Homarus americanus are classified as chelicerata and crustaceans, respectively. Their structurally well-characterized hemocyanins are composed of, in the case of E. californicum 24 subunits, and in the case of H. americanus 12 subunits. The role of protons as allosteric effectors of the oxygen binding was analysed in terms of the nesting model, which assumes hierarchies of allosteric equilibria that are based on obvious structural hierarchies. For each hemocyanin, the smallest structural repeating unit, the 12-mer or the 6-mer, respectively, was regarded as the "allosteric unit". Two allosteric units are allosterically coupled within the native molecules. The analysis revealed that in accordance with the postulations of the classical Monod-Wyman-Changeux model protons as allosteric effectors do not change the oxygen affinities of the four postulated conformations, but influence the allosteric equilibria between them at two different hierarchical levels. Model-independent determination of the affinity constants for the binding of the first and the last oxygen molecule to the native hemocyanins and to the isolated half-molecules confirmed the affinities calculated according to the nesting model. The stepwise establishment of new conformations during the assembly process from monomers to the structurally identical repeating unit and further on to the native molecule is shown. Possible physiological advantages of allosterically coupled allosteric units in contrast to allosterically uncoupled ones are thought to be (1) the option to regulate oxygen binding on different levels of structural hierarchy and (2) the increase of the oxygen-carrying capacity.     

The structure of the Escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates. The subunit folding and the dimeric basic structural unit are remarkably the same for available structures but, depending on species, dimers self-associate to form hexamers or tetramers. The crystal structure of the Escherichia coli NDPK reveals a new tetrameric quaternary structure for this protein family. The two tetramers differ by the relative orientation of interacting dimers, which face either the convex or the concave side of their central sheet as in either Myxococcus xanthus (type I) or E. coli (type II), respectively. In the type II tetramer, the subunits interact by a new interface harboring a zone called the Kpn loop as in hexamers, but by the opposite face of this loop. The evolutionary conservation of the interface residues indicates that this new quaternary structure seems to be the most frequent assembly mode in bacterial tetrameric NDP kinases.     

The allosteric effector l-lactate induces a conformational change of 2x6-meric lobster hemocyanin in the oxy state as revealed by small angle x-ray scattering

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.     

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.     

Structural studies on a low oxygen affinity hemoglobin from mammalian species: Sheep (Ovis aries)

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express ‘high’ and ‘low’ oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7 Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the “joint region”, shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1β2/α2β1 interfaces of sheep metHb structure. In addition, the constrained β subunits heme environment and the contraction of N-termini and A-helices of β subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.