Global allostery model of hemoglobin. Modulation of O(2) affinity,cooperativity, and Bohr effect by heterotropic allosteric effectors

The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin.     

How does hemoglobin generate such diverse functionality of physiological relevance?

The absolute values of the O₂-affinities (P₅₀, K_low, and K_high) of hemoglobin (Hb) are regulated neither by changes in the static T-/R-quaternary and associated tertiary structures nor the ligation states. They are pre-determined and regulated by the extrinsic environmental factors such as pH, buffers, and heterotropic effectors. The effect and role of O₂ on Hb are reversibly to drive the structural allosteric equilibrium between the T(deoxy)- and R(oxy)-Hb toward R(oxy)-Hb (the structural allostery). R(oxy)-Hb has a higher O₂-affinity (K_high) relative to that (K_low) of the T(deoxy)-Hb (K_high > K_low) under any fixed environmental conditions. The apparent O₂-affinity of Hb is high, as the globin matrix interferes with the dissociation process of O₂, forcing the dissociated O₂ geminately to re-bind to the heme Fe. This artificially increases [oxy-Hb] and concomitantly decreases [deoxy-Hb], leading to the apparent increases of the O₂-affinity of Hb. The effector-linked high-frequency thermal fluctuations of the globin matrix act as a gating mechanism to modulate such physical, energetic, and kinetic barriers to enhance the dissociation process of O₂, resulted in increases in [deoxy-Hb] and concomitant decrease in [oxy-Hb], leading to apparent reductions of the O₂-affinity of Hb (the entropic allostery). The heme in Hb is simply a low-affinity O₂-trap, the coordination structure of which is not altered by static T-/R-quaternary and associated tertiary structural changes of Hb. Thus, heterotrophic effectors are the signal molecule, which acts as a functional link between these two allosteries and generates the diverse functionality of Hb of physiological relevance. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Molecular dynamics simulations of hemoglobin A in different states and bound to DPG: effector-linked perturbation of tertiary conformations and HbA concerted dynamics

Recent functional studies reported on human adult hemoglobin (HbA) show that heterotropic effector-linked tertiary structural changes are primarily responsible for modulating the oxygen affinity of hemoglobin. We present the results of 6-ns molecular dynamics simulations performed to gain insights into the dynamical and structural details of these effector-linked tertiary changes. All-atom simulations were carried out on a series of models generated for T- and R-state HbA, and for 2,3-diphosphoglycerate-bound models. Cross-correlation analyses identify both intra- and intersubunit correlated motions that are perturbed by the presence of the effector. Principal components analysis was used to decompose the covariance matrix extracted from the simulations and reconstruct the trajectories along the principal coordinates representative of functionally important collective motions. It is found that HbA in both quaternary states exists as ensembles of tertiary conformations that introduce dynamic heterogeneity in the protein. 2,3-Diphosphoglycerate induces significant perturbations in the fluctuations of both HbA states that translate into the protein visiting different tertiary conformations within each quaternary state. The analysis reveals that the presence of the effector affects the most important components of HbA motions and that heterotropic effectors modify the overall dynamics of the quaternary equilibrium via tertiary changes occurring in regions where conserved functionally significant residues are located, namely in the loop regions between helices C and E, E and F, and F and G, and in concerted helix motions. The changes are not apparent when comparing the available x-ray crystal structures in the presence and absence of effector, but are striking when comparing the respective dynamic tertiary conformations of the R and T tetramers.     

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.     

Correlating allostery with rigidity

Allosteric proteins demonstrate the phenomenon of a ligand binding to a protein at a regulatory or effector site and thereby changing the chemical affinity of the catalytic site. As such, allostery is extremely important biologically as a regulatory mechanism for molecular concentrations in many cellular processes. One particularly interesting feature of allostery is that often the catalytic and effector sites are separated by a large distance. Structural comparisons of allosteric proteins resolved in both inactive and active states indicate that a variety of structural rearrangement and changes in motions may contribute to general allosteric behavior. In general it is expected that the coupling of catalytic and regulatory sites is responsible for allosteric behavior. We utilize a novel examination of allostery using rigidity analysis of the underlying graph of the protein structures. Our results indicate a general global change in rigidity associated with allosteric transitions where the R state is more rigid than the T state. A set of allosteric proteins with heterotropic interactions is used to test the hypothesis that catalytic and effector sites are structurally coupled. Observation of a rigid path connecting the effector and catalytic sites in 68.75% of the structures points to rigidity as a means by which the distal sites communicate with each other and so contribute to allosteric regulation. Thus structural rigidity is shown to be a fundamental underlying property that promotes cooperativity and non-locality seen in allostery.     

Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

Trapping quaternary structures of hemoglobin in single crystals or by encapsulation in silica gels has provided a demanding set of data to test statistical mechanical models of allostery. In this work, we compare the results of those experiments with predictions of the four major allosteric models for hemoglobin: the quaternary two-state model of Monod, Wyman, and Changeux; the tertiary two-state model of Henry et al., which is the simplest extension of the Monod-Wyman-Changeux model to include pre-equilibria of tertiary as well as quaternary conformations; the structure-based model of Szabo and Karplus; and the modification of the latter model by Lee and Karplus. We show that only the tertiary two-state model can provide a near quantitative explanation of the single-crystal and gel experimental results.     

High pressure reveals that the stability of interdimeric contacts in the R- and T-state of HbA is influenced by allosteric effectors: Insights from computational simulations

The molecular details of the mechanism of action of allosteric effectors on hemoglobin oxygen affinity are not clearly understood. The global allostery model proposed by Yonetani et al. suggests that the binding of allosteric effectors can take place both in the R and T states and that they influence oxygen affinity through inducing global tertiary changes in the subunits. Recently published high pressure studies yielded dissociation constants at atmospheric pressure that showed a stabilizing effect of heterotropic allosteric effectors on the dimer interface in the R state, and a more pronounced destabilizing effect in a T state model. In the present work, we report on computational modeling used to interpret the high pressure experimental data. We show structural changes in the hemoglobin interdimeric interfaces, indicative of a global tertiary structural change induced by the binding of allosteric effectors. We also show that the number of water molecules bound at the interface is significantly influenced by binding effectors in the T state in accordance with the experimental data. Our results suggest that the binding of effectors at definite sites leads to tertiary changes that propagate to the interfaces and results in overall structural re-organizations.     

Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.     

Ligand binding and allostery can emerge simultaneously

A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 beta-lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has approximately 10(6) M(-1) affinity for Zn(2+), a property that neither of the parental proteins possesses. Furthermore, Zn(2+) is a negative effector that noncompetitively switches off beta-lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn(2+)-binding site does not involve a histidine or a cysteine, which is atypical of natural Zn(2+)-binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.     

Allostery in GPCRs: 'MWC' revisited

G protein-coupled receptors (GPCRs) constitute the largest family of receptors in the genome and are the targets for at least 30% of current medicines. In recent years, there has been a dramatic increase in the discovery of allosteric modulators of GPCR activity and a growing appreciation of the diverse modes by which GPCRs can be regulated by both orthosteric and allosteric ligands. Interestingly, some of the contemporary views of GPCR function reflect characteristics that are shared by prototypical allosteric proteins, as encompassed in the classic Monod-Wyman-Changeux (MWC) model initially proposed for enzymes and subsequently extended to other protein families. In this review, we revisit the MWC model in the context of emerging structural, functional and operational data on GPCR allostery.     

Effector-induced structural fluctuation regulates the ligand affinity of an allosteric protein: binding of inositol hexaphosphate has distinct dynamic consequences for the T and R states of hemoglobin

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.     

Allosteric effectors influence the tetramer stability of both R- and T-states of hemoglobin A

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl-, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (alphaFeO2)2-(betaZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70-200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl-. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.     

High and low oxygen affinity conformations of T state hemoglobin

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.     

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.     

Description of hemoglobin oxygenation under universal solution conditions by a global allostery model with a single adjustable parameter

The Monod-Wyman-Changeux allosteric model parameters evaluated from accurate oxygen equilibrium curves (OECs) of hemoglobin that were measured in an extremely wide range of structural constraints, imposed by allosteric effectors, yielded a closed circle when log K(T) and log K(R) were plotted against log L(0) and log L(4), respectively, showing novel phenomena that L(0) and L(4) have a maximal value and a minimal value, respectively, and K(T) and K(R) vary by more than three orders of magnitude. These phenomena were successfully described by a global allostery model, which mathematically keeps the frame work of the MWC model, but allows that K(T) under a set of solution conditions becomes larger than K(R) under another set of solution conditions and postulates that a representative allosteric effector binds to both the T and R states with a lower affinity but with a larger stoichiometry for the R state than for the T state. Thus, this global model can describe any given OEC measured under universal solution conditions with the single adjustable parameter, the concentration of the representative effector.     

Liganded hemoglobin structural perturbations by the allosteric effector L35

Effector binding to liganded hemoglobin (Hb) provides a new understanding of structural determinants of Hb function. L35, a bezafibrate-related compound, is one of the more potent synthetic regulators of Hb oxygen (O(2)) affinity. In the presence of inositol hexaphosphate and bezafibrate (or derivatives), liganded Hb at low pH (pH approximately 6.5) exhibits extremely low O(2) affinity and very low cooperativity. In this study, the nature of L35 binding to COHbA at pH 6.35, an altered R-state, is presented. Solution-active site-specific spectroscopic probings by front-face fluorescence and circular dichroism reveal that L35 induces a global heterogeneous conformation in COHbA at pH 6.35 that includes: a T-like structural feature at the alpha1beta2 interface; an R-like structural feature within the heme environment; and an intermediate-like state at the central cavity. These long-range structural perturbations appear to stem from L35 binding to two classes of binding sites: the central cavity (primarily at the alphaalpha cleft) and the surface. These results indicate that L35 induces an allosteric transition species, characterized by domain-specific tertiary and quaternary-like conformation within a global R-quaternary structure.