Hemoglobin Allostery Research Articles

A retrospective on statistical mechanical models for hemoglobin allostery.

Understanding allosteric interactions in proteins has become one of the major research areas in protein science. The original aim of the famous theoretical model of Monod, Wyman, and Changeux (MWC) was to explain the regulation of enzymatic activity in biochemical pathways. However, its first successful quantitative application was to explain cooperative oxygen binding by hemoglobin, often called the "hydrogen molecule of biology." The combination of its original application and the enormous amount of research on hemoglobin has made it the paradigm for studies of allostery, especially for multi-subunit proteins, and for the development of statistical mechanical models to describe how structure determines function. This article is a historical account of the development of statistical mechanical models for hemoglobin to explain both the cooperative binding of oxygen (called homotropic effects by MWC) and how oxygen binding is affected by ligands that bind distant from the heme oxygen binding site (called heterotropic allosteric effects by MWC). This account makes clear the many remaining challenges for describing the relationship of structure to function for hemoglobin in terms of a satisfactory statistical mechanical model.

Modulating hemoglobin allostery for treatment of sickle cell disease: current progress and intellectual property

ABSTRACT Introduction Sickle cell disease (SCD) is a debilitating inherited disorder that affects millions worldwide. Four novel SCD therapeutics have been approved, including the hemoglobin (Hb) modulator Voxelotor. Areas Covered This review provides an overview of discovery efforts toward modulating Hb allosteric behavior as a treatment for SCD, with a focus on aromatic aldehydes that increase Hb oxygen affinity to prevent the primary pathophysiology of hypoxia-induce erythrocyte sickling. Expert Opinion The quest to develop small molecules, especially aromatic aldehydes, to modulate Hb allosteric properties for SCD began in the 1970s; however, early promise was dogged by concerns that stalled support for research efforts. Persistent efforts eventually culminated in the discovery of the anti-sickling agent 5-HMF in the 2000s, and reinvigorated interest that led to the discovery of vanillin analogs, including Voxelotor, the first FDA approved Hb modulator for the treatment of SCD. With burgeoning interest in the field of Hb modulation, there is a growing landscape of intellectual property, including drug candidates at various stages of preclinical and clinical investigations. Hb modulators could provide not only the best chance for a highly effective oral therapy for SCD, especially in the under-developed world, but also a way to treat a variety of other human conditions.

Allosteric transitions in hemoglobin revisited

BackgroundHuman hemoglobin is an allosteric protein that exerts exquisite control over ligand binding through large-scale conformational changes. The two-state model without intermediates offers a simple qualitative description of the allosteric behavior of hemoglobin, as presented in textbooks. However, there is renewed interest in this topic due to recent experimental breakthroughs that show how hemoglobin actually undergoes conformational transitions in response to environmental changes. Scope of reviewI review the current understanding of hemoglobin structure-function relationships revealed by recent discoveries. A unique single crystal, in which three protein molecules are allowed to express a whole range of quaternary structures, helped to reveal the detailed transition pathway including various intermediate forms. I also discuss the potential of single-molecule techniques that are currently under examination. Major conclusionsNew crystallographic approaches reveal that the hemoglobin allosteric transition involves population shifts in multiple quaternary conformers rather than a simple two-state switch, and that coexisting individual conformers may have disproportionate effects on the apparent O2 affinity of hemoglobin. General significanceThese approaches provide a further level of complexity on the textbook statement of hemoglobin allostery, highlighting the relevance of conformational distributions in controlling the function and regulation of allosteric proteins.

A new picture of hemoglobin allostery

A new picture of hemoglobin allostery

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin.

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and β chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and β (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and β subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO.

New look at hemoglobin allostery.

New look at hemoglobin allostery.

A Carbon Monoxide Sensing Film Based on Hemoglobin Allostery

Carbon monoxide (CO) is a gaseous mediator, which is generated via anenzymatic reaction of heme oxygenase, and it plays physiological roles to regulate cellular respiration and blood flow in the liver. The concentration and distribution of CO molecules in the living body is unknown owing to a lack of a suitable technique for measuring them in vivo. A needle-type CO sensor has been used for bioinstrumentation, but it is inappropriate for implantation in vivo and long-term monitoring. We developed a CO sensor sheet based on hemoglobin (Hb) allostery, as Hb undergoes a conformational change on CO binding. Hb was extracted from mice blood and mixed with agarose gel with a reducer to stabilize deoxy-Hb in the gel. CO-releasing molecules (CORM) were used to mimic CO-generating tissue, and the sensitivity of the Hb gel could be regulated by Hb concentration. We defined the CO–Hb index, an absorbance ratio at 539 and 557 nm, to estimate the accumulation of captured CO in the gel. It correlatively increased with CORM dose, indicating that gel-embedded Hb underwent a conformational change on CO binding, thereby acting as a CO sensor. We subsequently used the Hb-sensor sheet for two-dimensional imaging of CO distribution. CORM-containing gels with different sizes and doses were layered on this sheet. Size- and dose-dependent CO distribution was visualized by scanning the CO-Hb index in the sheet. Our Hb-based CO sensor sheet is composed of biocompatible materials and can be applied to detect low-level CO sources in the living body.

Hemoglobin Allostery: New Views on Old Players

Proteins are dynamic molecular machines whose structure and function are modulated by environmental perturbations and natural selection. Allosteric regulation, discovered in 1963 as a novel molecular mechanism of enzymatic adaptation [Monod, Changeux & Jacob (1963). J. Mol. Biol.6, 306–329], seems to be the leit motiv of enzymes and metabolic pathways, enabling fine and quick responses toward external perturbations. Hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the paradigmatic system to test the validity of the conformational selection mechanism, the conceptual innovation introduced by Monod, Wyman and Changeux. We present hereby the results of a comparative analysis of structure, function and thermodynamics of two extensively investigated hemoglobins: human HbA and trout HbI. They represent a unique and challenging comparison to test the general validity of the stereochemical model proposed by Perutz. Indeed both proteins are ideal for the purpose being very similar yet very different. In fact, T-HbI is a low-ligand-affinity cooperative tetrameric Hb, insensitive to all allosteric effectors. This remarkable feature, besides being physiologically sound, supports the stereochemical model, given that the six residues identified in HbA as responsible for the Bohr and the 2,3-di-phosphoglycerate effects are all mutated. Comparison of the three-dimensional structures of HbA and T-HbI allows unveiling the molecular mechanism whereby the latter has a lower O2 affinity. Moreover, the energetic balance sheet shows that the salt bridges breaking upon allosteric quaternary transition are important yet insufficient to account for the free energy of heme–heme interactions in both hemoglobins.

Structural Dynamics of Clamshell Rotation during the Incipient Relaxation Process of Photodissociated Carbonmonoxy Myoglobin: Statistical Analysis by the Perturbation Ensemble Method

The structural dynamics of the clamshell rotation of photodissociated carbonmonoxy myoglobin, which is expected to be important for hemoglobin allostery, is investigated by the perturbation ensemble method. In this method, many pairs of perturbed and unperturbed molecular dynamics trajectories are ensemble-averaged to cancel out thermal noises and to detect subtle changes. The number of MD trajectory pairs, in this work 2000 pairs, should be determined to obtain physical properties of interest with statistically meaningful precisions. The calculated structural changes after 20 ps of the photodissociation are consistent with those by time-resolved X-ray diffraction at 100 ps delay time. In the heme proximal side region including the F and H helices, both helices displaced in the proximal direction. Meanwhile, in the heme distal side region including E and A helices, both helices moved toward the heme group after photodissociation. These proximal and distal side displacements occur on a fast time scale (almost complete within 3 ps) and are consistent with the clamshell rotation. Moreover, it was found that the ensemble-averaged structural dynamics of the photodissociated MbCO is independent of the amount of initial excess vibrational energy of the heme, or the difference of excitation photon wavelength. These results provide atomistic details on the functionally important dynamics of the clamshell rotation. Application of the present methodology to Hb will give new insight into the incipient stereochemical mechanism of hemoglobin allostery.

2P104 ヘモグロビンのアロステリーはMWCモデルと調和できるか?(ヘム蛋白質,第48回日本生物物理学会年会)

2P104 ヘモグロビンのアロステリーはMWCモデルと調和できるか?(ヘム蛋白質,第48回日本生物物理学会年会)

Roles of Hemoglobin Allostery in Hypoxia-induced Metabolic Alterations in Erythrocytes: SIMULATION AND ITS VERIFICATION BY METABOLOME ANALYSIS

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.

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.

Modeling of Ligation-Induced Helix/Loop Displacements in Myoglobin: Toward an Understanding of Hemoglobin Allostery

Combining quantum and molecular mechanics (QM/MM) methods and protein structure prediction algorithms, helix and loop movements are computed along the pathway of CO dissociation from myoglobin (Mb). The results are compared with high-resolution crystallographic data using sequence-displacement graphs. These graphs provide an unbiased method for evaluating main-chain segmental motions; they resolve an apparent disagreement between two sets of high-resolution crystal structures for MbCO and deoxyMb. The QM/MM modeling of the CO deligation reproduces the experimentally observed spin states and photodissociated crystal structure. The principal effect of CO dissociation is shown to be a concerted rotation of the E and F helices, which hold the heme like a clamshell. The rotation is a response to deligation forces, which impel the F helix away from the heme because of the Fe spin conversion, and which allow the E helix to collapse toward the heme as nonbonded contacts on the distal side are relieved. Additional helix and loop displacements stem from these primary events. In particular, the CD loop is found to be repositioned as a result of steric interactions with the water molecule that becomes H-bonded to the distal histidine in deoxyMb. A similar EF rotation and CD loop displacement are proposed to be the first steps along the allosteric pathway from the R to the T state in hemoglobin.

The molecular code for hemoglobin allostery revealed by linking the thermodynamics and kinetics of quaternary structural change. 1. Microstate linear free energy relations.

A novel model linking the thermodynamics and kinetics of hemoglobin's allosteric (R --> T) and ligand binding reactions is applied to photolysis data for human HbCO. To describe hemoglobin's kinetics at the microscopic level of structural transitions and ligand-binding events for individual [ij]-ligation microstates ((ij)R --> (ij)T, (ij)R + CO --> ((i)(+1))(k)R, and (ij)T + CO --> ((i)(+1))(k)T), the model calculates activation energies, (ij)DeltaG(++), from previously measured cooperative free energies of the equilibrium microstates (Huang, Y., and Ackers, G. K. (1996) Biochemistry 35, 704-718) by using linear free energy relations ((ij)DeltaG(++) - (01)DeltaG(++) = alpha[(ij)DeltaG - (01)DeltaG], where the parameter alpha, describing the variation of activation energy with reaction energy perturbation, can depend on the natures of both the reaction and the perturbation). The alpha value measured here for the allosteric dynamics, 0.21 +/- 0.03, corresponds closely to values observed previously, strongly suggesting that the thermodynamic microstate energies directly underlie the allosteric kinetics (as opposed to the alpha((ij)DeltaG(RT)) serving merely as arbitrary fitting parameters). Besides systematizing the study of hemoglobin kinetics, the utility of the microstate linear free energy model lies in the ability to test microscopic aspects of allosteric dynamics such as the "symmetry rule" for quaternary change deduced previously from thermodynamic evidence (Ackers, G. K., et al. (1992) Science 255, 54-63). Reflecting a remarkably detailed correspondence between thermodynamics and kinetics, we find that a kinetic model that includes the large free energy splitting between doubly ligated T microstates implied by the symmetry rule fits the data significantly better than one that does not.

Nuclear Magnetic Resonance Spectroscopy in the Study of Hemoglobin Cooperativity

Bacterial expression systems have greatly advanced NMR-based studies of Hb A and understanding of the relationship between structure and function in Hb. These advances result largely from the ability to isotopically label HbA for high-resolution heteronuclear NMR studies, literally opening up new dimensions in the NMR of hemoglobin, permitting extensive resonance assignments to be made, providing information about dynamics and hydrogen bonding, and yielding precise information regarding quaternary structure in solution. These expression systems also permit site-specific substitutions to be introduced to the Hb tetramer. NMR spectroscopy provides a powerful tool for quickly determining the effects of site-specific substitutions on the structure of the heme pocket, on site-specific distal ligand binding, on subunit interface structure and dynamics, and on the overall quaternary structure in solution.One area where NMR spectroscopy is likely to further advance the understanding of hemoglobin allostery is conformational dynamics. A few sites at the α 1 β 2 subunit interface appear to be quite dynamic over a range of time scales, and these dynamics appear to be coupled to distal ligand binding and quaternary structure. By fully characterizing the range of dynamics of different forms of Hb, NMR spectroscopy provides a picture of hemoglobin allostery that is considerably more detailed than that provided by the static structures obtained from X-ray crystallography. Another area where NMR spectroscopy is likely to advance the understanding of structure and function in hemoglobin is in determination of solution quaternary structure of the T state, using the residual dipolar coupling method. Residual dipolar coupling methods can determine whether deoxy-Hb A in solution conforms well to the crystallographically observed T-quaternary structure, or whether additional structures are also present, as in distally ligated HbCO A. Although hemoglobin has been studied with great intensity by a wide variety of methods over the last half-century, many of the molecular details that control allostery are still unknown. Experimental results on rHbs derived from NMR, equilibrium, and kinetic studies of ligand binding clearly indicate that hemoglobin is a rather flexible protein, and that its conformation can adapt to a variety of perturbations, for example, amino acid substitutions, binding of ligands, and allosteric effectors. These results also indicate that allosteric interactions in Hb A involve multiple pathways of signal transmission.

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

Hydrogen bonding to Trp beta37 is the first step in a compound pathway for hemoglobin allostery.

Human hemoglobin is widely thought to change from the R to the T quaternary structure in a single rate process requiring tens of microseconds. Here we present kinetic evidence that the R --> T allosteric pathway in hemoglobin requires more than one step. We use magnetic circular dichroism (MCD) spectroscopy of the aromatic amino acid bands to show that formation of a tryptophan-aspartate hydrogen bond in the hinge region of the dimer-dimer interface is part of an obligatory R --> T step proceeding more than a factor of 10 faster than the kinetic step previously identified in heme-band absorption studies.

Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery.

The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.

Trans-substitution of the proximal hydrogen bond in myoglobin: II. Energetics, functional consequences, and implications for hemoglobin allostery.

The trans-substituted histidine to glycine mutant of sperm whale myoglobin (H93G Mb) is used to study energetics of proximal hydrogen bonding, proximal ligand-heme interactions, and coupling to distal ligand binding. Comparison of mono- and dimethylimidazole structural isomers shows that the hydrogen bond between the proximal ligand and the neighboring Ser92 hydroxyl (position F7) is stabilizing. The range of hydrogen bond stabilities measured here for different distal ligand complexes ranges from -0.7 kcal/mol (monomethylimidazole isomers to MbCO) to -4.1 kcal/mol (dimethylimidazole isomers to MbCN). This range of hydrogen bond stabilities, which is similar to that seen in protein mutagenesis unfolding studies, demonstrates the high sensitivity of the hydrogen bond to modest structural perturbations. The degree to which the 2-methyl group destabilizes proximal ligand binding is found to depend inversely on the total electronic spin. For monomethylimidazole proximal ligands, distal ligand binding weakens the proximal hydrogen bond compared to deoxyMb. Surprisingly, this trend is largely reversed for the dimethylimidazole proximal ligands. These results demonstrate strong coupling between the proximal protein matrix and distal ligand binding. These results provide an explanation for the strong avoidance of hydrogen bonding residues at position F7 in hemoglobin sequences.

Kinetics of hemoglobin allostery from time-resolved UV resonance Raman spectroscopy: effect of a chemical cross-link

Three kinetic phases along the allosteric reaction path of hemoglobin were determined using ultraviolet resonance Raman (UVRR) difference signals associated with tyrosine and tryptophan residues. The CO adduct was photolyzed with saturating 419 nm pump pulses, and UVRR spectra were generated at a series of time delays with 229 nm probe pulses. Pump and probe pulses were generated by a pair of 1 kHz, 20 ns Nd : YLF-pumped Ti : sapphire lasers, whose outputs were frequency doubled (pump) and quadrupled (probe). Difference spectra obtained with this system are of better quality than those reported previously, and lead to a better defined time course of the spectral changes associated with the protein motions. Fitting of the time course to successive exponential produces time constants of 0.03, 0.7 and 16 µs for the intermediates Rdeoxy, S and T′, in good agreement with previous estimates. A chemically modified hemoglobin, α αHb, was also examined. α αHb, which is prepared by cross-linking the α-chains with bis(3,5-dibromosalicyl) fumarate, is under study as a blood substitute. The time course for α αHb was similar to that of unmodified Hb, and the time constants were 0.04, 0.35 and 20 µs. The first and last of the kinetic phases are essentially unaltered, but the second phase is accelerated by a factor of two. Thus the cross-link speeds up the Rdeoxy to S transition, which is proposed to involve re-formation of interhelical H-bonds that are broken in the Rdeoxy intermediates via repositioning of the N- and C-terminal helices A and H. This transition may be guided by the cross-link which connects the two α-chain G helices in the T conformation. Copyright © 2000 John Wiley & Sons, Ltd.