REVIEW ARTICLE
Variations on the theme: allosteric control in hemoglobin Maurizio Brunori Istituto Pasteur – Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Italy
Keywords allosteric models; evolution of hemoglobin; heterotropic effects; physiological requirements; structure and function Correspondence M. Brunori, Dipartimento di Scienze Biochimiche, Sapienza University of Rome, P. le Aldo Moro 5, 00185 Rome, Italy Fax: +39 06 49910717 Tel: +39 06 4450291 E-mail: [email protected]
Conformational selection between pre-existing structural states of an oligomeric protein was the conceptual step behind the formulation of Monod– Wyman–Changeux (MWC) allosteric theory. Variations on the basic theme of allosteric control are briefly illustrated in this paper by reference to some hemoglobins from different species whose functional properties were found to respond to specific physiological requirements. In my opinion the enormous success of the allosteric theory may be attributed not only to its efficiency in accounting for data and its formal mathematical elegance, but also because the selective mechanism conforms to the founding concept of Darwinian evolution.
(Received 22 July 2013, revised 10 September 2013, accepted 23 September 2013) doi:10.1111/febs.12586
Forward According to the records, the term ‘allosteric inhibition’ was coined in 1961 by Jacques Monod and Francßois Jacob in a written comment to the paper presented by Jean-Pierre Changeux at the Cold Spring Harbor Symposium on Quantitative Biology; the history of the events that led to the fundamental findings and the conception of the theoretical model have been presented by Changeux himself ([1,2] and references therein). After the first full paper that appeared in 1963 [3], the theory was elaborated and formalized in the paper by Monod, Wyman and Changeux [4] published in the Journal of Molecular Biology in May 1965. This seminal work proved to be most inspiring, a remarkable success for a theoretical paper that over the years has been extensively cited. An interesting historical account of the scientific interactions between Jacques Monod and Jeffries Wyman (from January 1963 to January 1965) and the chronology of the events was presented by Henri Buc [5] at a meeting commemorating the 40th
anniversary of the Monod–Wyman–Changeux (MWC) model, held at the Accademia Nazionale dei Lincei in Rome in May 2005. It is not difficult to understand why hemoglobin (Hb) became the paradigmatic protein to illustrate the theory of allosteric control. In the early 1960s a great deal of innovative quantitative information was already available and carefully analyzed: the 3D structure of Hb had been solved by Max Perutz et al. [6,7] and shown to be different in the oxy and deoxy states; cooperative O2 binding and the Bohr effect had been extensively studied [8,9]; several natural mutants of human Hb (including some pathological variants) and the Hbs from many different species of vertebrates had been investigated [10]; and much more … [11,12]. In 1951 a seminal idea had been conceived by Wyman and Allen [13] as a result of an analysis of quantitative data on the Bohr effect, much before any knowledge of the structure of the protein became available.
Abbreviations BPG, 2,3-bisphosphoglycerate; Hb, hemoglobin.
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Interpreting the Bohr effect, Wyman and Allen wrote: ‘the reason why certain acid groups are affected by oxygenation is simply the alteration of their position and environment which results from the change of configuration of the hemoglobin molecule as a whole accompanying oxygenation, …’. It was realized that proteins with their complexity of possible configurations (nowadays called the ‘conformational landscape’) would be fitted to play the role of an enzyme, a prophetic idea that years later justified the promotion of Hb to the rank of an honorary enzyme: ‘if we are prepared to accept hemoglobin as an enzyme, its behavior might give us a hint as to the kind of process to be looked for in enzymes more generally’ [13]. In 1966 Koshland, Nemethy and Filmer [14] published an alternative model based on the concept of induced fit, whereby binding of a substrate induces a conformational change in the relevant subunit of the oligomeric enzyme. Thus, contrary to the basic concept of allostery, induced fit is a sequential model with population of the reaction intermediate states. The KNF model has been extensively applied especially in molecular enzymology, being originally conceived to account for enzyme specificity. It would demand a whole paper to highlight the relative merits of the MWC and KNF models in describing the behavior of Hb; and clearly this is not the place. Still it seemed only fair to recall the seminal contribution of Dan Koshland and collaborators [14,15], generally illustrated in all biochemistry textbooks. In this short account I shall not deal in any detail with the paradigmatic case of human HbA, probably the most extensively studied protein, but will highlight the general consistency with the allosteric model of the peculiar extreme behavior of some interesting Hbs from fish and birds.
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emerged as the common answer in vertebrates. All of them synthesize a2b2 tetrameric Hb as the canonical O2 carrier, characterized by allosteric conformational control to maximize efficiency in delivery. Allostery is the general mechanism adopted in the evolution of vertebrates to provide all cells in the body with sufficient O2 for energy production. The large variability in O2 demands under very different environmental conditions (changes in temperature, pressure, pH, O2 concentration etc.) has been confronted with peculiar molecular responses [16] adapting the efficiency of O2 delivery by Hb to respond to specific and variable physiological demands. Particularly interesting and challenging has been the correlation between the physiological needs in the context of a specific environment and the modifications of the structural and functional properties of Hb brought about by mutation and selection [16], maintaining the mechanism of allosteric control to optimize O2 delivery. In Wyman’s words [17]: ‘The theme is hemoglobin; the variations, the differences in its properties and behavior which have been developed in the course of evolution to meet special requirements of different animal forms’. Extending the study of physiology and biochemistry of blood to many species relies on nature doing the work of chemical modification. The scientific expedition to the Amazon in 1976 was justified by the curiosity to investigate the exotic species of fish living in that mother of rivers. This paper written for the jubilee of the allosteric theory highlights a few paradigmatic examples of fascinating molecular adaptation to peculiar physiological requirements as observed in different animal species, and their consistency with the basic features of the MWC theory.
The energy level diagram The demand for an oxygen carrier A dramatic revolution in biological evolution has been the transition from a reductive to an oxidizing atmosphere with a large increase in the partial pressure of O2 produced by photosynthesis. The opportunity for more efficient energy production, that eventually culminated in the mitochondrial oxidative phosphorylation, made the difference. The emergence of vertebrates and the general increase in body mass demanded huge energy production/ATP synthesis and thereby some appropriate devices to make O2 available to every cell of every organ in the body, given that gas diffusion proved inadequate for large multicellular organisms. Circulation of blood and the synthesis of a super-specialized carrier cell, the erythrocyte, have 634
The essential framework of the allosteric theory is depicted in Fig. 1A in terms of a diagram indicating the energy levels of the two quaternary allosteric states of a tetramer with four chemically identical subunits, each binding a ligand [18,19]. In going from top to bottom, the scheme highlights the increasing number of ligands bound from zero (deoxyHb) to four (fully oxygenated Hb). In the absence of ligand, the population ratio between states is expressed by the fundamental constant L0 = [T0]/[R0], which for HbA may vary between 103 and 107 (depending on experimental conditions). The a and b subunits in tetrameric a2b2Hb are characterized by subtle differences in tertiary structure that may play a crucial role in functional control. FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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A
B
Fig. 1. Energetics of the MWC model, and the Hill plot. (A) Energy level diagram indicating the two different quaternary states (squares and circles) and the 10 ligation species (T0 to T4 and R0 to R4). The fundamental equilibrium constants of the MWC model [4] are shown: KT and KR, the oxygen dissociation constants of the two allosteric states T (tense) and R (relaxed); and L0, the population ratio of the two states in the fully deoxygenated tetrameric Hb. The implicit assumption is that for a fully concerted quaternary transition the switch-over point will be at the level T2– R2 in the case of a symmetric binding curve. Typical values of the parameters for human HbA at neutral pH and 20 °C are ~ L0 = [T0]/ [R0] = 105 and c = KR/KT = 0.01. The allosteric equilibrium constant depends on the number of ligands bound, according to the equation Li = L0ci, with i from 0 to 4. (B) Hill plot of the oxygen equilibrium of horse Hb in 0.6 M phosphate buffer at pH 7.0 and 19 °C; different techniques were employed to acquire data in the different saturation ranges going from < 1% to ~ 99%. Total free energy of interaction DFi = 2.6 kcalmol 1 (heme); nH = 2.95. Data of RLJ Lyster as reported by Wyman [9], modified.
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The basic components of the model are (a) the presence of two structural states of the oligomer (R and T) that are populated at every level of saturation; and (b) the difference in ligand affinity between the two allosteric states, defined by the dissociation equilibrium constants KR and KT [4]. In the scheme of Fig. 1A, T0 is more populated than R0 and thus, despite its lower O2 affinity (KR 1000-fold, then binding may occur to the R-state albeit the least populated of species, and the Hill coefficient will tend to nH = 1. In addition, since all molecular species are always present at equilibrium, if L0 decreases considerably due (for example) to destabilization of the T-state, the binding will tend to occur preferentially by the R-state and thus O2 affinity will be high (approaching that of KR) and cooperativity low; and vice versa when the T-state is heavily stabilized, with a large increase in L0. These reciprocal relationships were analyzed in a formal paper by Wyman [21] and then elaborated by several other authors [12,18,22]. Figure 2 depicts the early analysis by Stuart Edelstein [22] who presented some of the experimental data available on HbA and some natural mutants, to highlight the correlation. Among the many examples arising from studies on human HbA under different conditions, a beautiful and particularly informative set of data on HbA trapped in crystals or in silica gel has been published by Mozzarelli et al. [23]. Trapping deoxyHb (in the T0-state) stabilizes a species that binds O2 with no apparent cooperativity and very low affinity, which has been interpreted as a single molecular state with characteristic tertiary and quaternary structures. This model has been extended to kinetics and is the basis for a novel attractive mechanism for human HbA, championed by Eaton et al. [24]. 635
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Fig. 2. Correlation between the Hill coefficient and the allosteric equilibrium constant. A classical demonstration of the dependence of the Hill coefficient nH (estimated between Y = 0.25 and Y = 0.75) on log L0 yielding a bell shaped curve, as predicted by the MWC model (see text). Numbers indicate data for HbA at two pH values; other data are for the isolated a and b subunits; (C) human Hb Chesapeake (Leu(FG4)92a?Arg); (S) stripped human HbA; (K) human Hb Kansas (Asn (G4)102b?Tyr) (from [22], with permission of SJ Edelstein).
The minimalist allosteric hemoglobin Figure 3 depicts the O2 binding isotherms of different Hbs that vary widely in their overall affinity and dependence of shape on pH and/or solvent composition. Human adult HbA (Fig. 3B) has been very extensively investigated [7,11,12,18,20,25] and is obviously the reference system throughout the field. The peculiar example depicted in Fig. 3A represents the O2 binding equilibrium of trout HbI, one of the components expressed in the blood of trout [26,27]. As may be seen from examination of the Hill plot, trout HbI is clearly cooperative with thermodynamic parameters [28] that are fairly similar to those of HbA under canonical experimental conditions, the maximum Hill coefficient being nH = 2.0–2.2 compared with nH = 2.8–3.0 for HbA and the overall free energy of interaction being (heme) compared with DFi = 2.2 kcalmol 1 DFi = 3.0 kcalmol 1 (heme) for HbA (see the distance between asymptotes). Trout HbI was shown to populate two different structural states based on a number of physical properties, the most direct being the 3D structure of the deoxy and CO derivatives solved by resolution. We have Tame et al. [29] at 2.2–2.4 A recently analyzed in depth the structural constraints and weak bonds that break in going from trout HbI deoxy (T0) to the CO bound state (R4) [30]. We concluded that the salt bridges broken during the allosteric transition [7,31] make a significant contribution to 636
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the difference in stability between the allosteric states, but cannot account alone for the overall free energy change calculated from the O2 binding isotherm. Several features of the structure–function relationships have been highlighted by Miele et al. [30]. The extraordinary feature of trout HbI is that the shape and position of the O2 binding curve is totally independent of solvent composition: no Bohr effect, no 2,3-bisphosphoglycerate (BPG) (or other phosphates) effect and indeed a very limited (almost zero) effect of temperature on p(O2)1/2 [26,27]. Moreover there is no evidence for ab subunit inequivalence; no significant dissociation into ab dimers even below micromolar concentration; and a switch-over point just above 2 ([30] and references therein). The dynamics of trout HbI have been extensively studied using time resolved laser spectroscopy [32] carried out over a wide range of fractional photolysis and temperatures from 2 to 65 °C. Results of this analysis compared with equilibrium data [28,33,34] confirmed the switch-over point to be close to doubly liganded, and the relaxation time for the R0?T0 transition around 20 ls, close to that determined for HbA by Sawicki and Gibson [35]. The lack of heterotropic effects (pH and phosphates) is quite peculiar. It was understood based on the findings obtained by amino acid sequence analysis [36]; as shown in Table 1, the residues that Perutz [7,25] proposed to be involved in the Bohr effect and in the organic phosphates effect are either absent or substituted by ineffective amino acids. This represents fully independent evidence in support of the classical assignment, and indeed almost proof for the mechanism of differential binding of protons and phosphate linked to oxygenation. In summary, trout HbI functional behavior is fully consistent with a ‘pure’ idealized MWC model, as depicted in Fig. 1A. However, the ligand-linked conformational equilibrium between R and T (Table 2) is totally insensitive to solvent composition, and thus efficiency of O2 delivery is solely based on homotropic interactions. The only variable affecting (somewhat) the relative population of states is temperature, with a standard enthalpy change for the T0R0 transition of ~ 30 kcalmol 1 of tetramer [28,34]. Since the population of the R0 state increases with temperature, L0 was calculated to be 1 at ~ 75–80 °C and naturally the Hill coefficient would tend to nH = 1. This prediction was confirmed by flash photolysis experiments on trout HbI-CO carried out up to ~ 72 °C [37]. Such a minimalist allosteric Hb responds effectively to tissue O2 demand but it is of limited latitude. It was proposed [27] that this type of Hb was selected for a FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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A
B
Fig. 3. Oxygen equilibrium curves of human and trout Hbs. Oxygen binding data are plotted in terms of a Hill plot, where Y is the fractional saturation with the ligand and pO2 is the oxygen partial pressure in the gas phase. (A) O2 binding curves of trout HbI at two pH values (indicated), in 0.2 M Bistris buffer at 4 °C (from [28], redrawn). (B) O2 binding curves of human HbA at different pH values (indicated) and 20 °C. In all cases 0.1 MCl was present; the curve on the right at pH 7.4 (triangles) contains also 2 mM inositol-hexaphosphate (from [20], redrawn). (C) O2 binding curves of trout HbIV at four different pH values (indicated), in 0.05 M Bistris buffer at 14 °C; a high pressure optical cell was employed for the very low affinity range (from [40], redrawn).
C
specific vital purpose expressed in fast swimming teleost fishes, while sluggish fish like carp do not express this type of Hb. In the case of hyperactive fish living in fast running waters, an Hb devoid of heterotropic control and representing a significant fraction (35%– 40%) of the total Hb in the blood fulfills the role of an emergency O2 supplier under critical metabolic demands related to strenuous exercise. In fact in hyperactive fish the production of lactic acid may be FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
sufficient to produce significant blood acidity which inhibits O2 supply to the tissues; the animal may suffer from internal asphyxia. The case of catostomid fish studied by Dennis Powers [38] is a particularly attractive example, given that members belonging to the subgenus Catostomus that live in fast moving waters express trout HbI-like carriers, whereas members of another subgenus (Pantosteus) living preferentially in pools do not. 637
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Table 1. Comparison between human HbA and trout HbI.
a a b b b b
subunit subunit subunit subunit subunit subunit
Human HbA
Trout HbI
Heterotropic ligand
Val1 (NH1) His122 (H5) His2 (NH2) Lys82 (EF6) His143 (H21) His146 (HC3)
Ac-Ser1 His123 Glu2 Leu82 Ser143 Phe146
Proton (Bohr effect) Proton (Bohr effect) BPG BPG BPG Proton (Bohr effect)
Amino acid substitutions directly linked to heterotropic allosteric effects; the topological positions are in parentheses.
Table 2. Allosteric equilibrium constant L0 for human and trout hemoglobins, at different pH values and 23–25 °C. L0 ([T0]/[R0]) pH 7.4 Trout HbI Human HbA Trout HbIV
3
3 x 10 8.7 x 104 4 x 103
pH 6.5 3
3 x 10 6.2 x 105 1 x 105
pH 6.0 3 x 103 3 x 106
Data from [20] and [28].
Extreme heterotropic control The O2 binding curves depicted in Fig. 3C are representative of the peculiar functional behavior common to all teleost fish Hbs, as reported long ago by Root [39]. In the case of trout, approximately 60% of the total Hb is characterized by such an extreme pH dependence of the shape and overall affinity [26]; given the very low affinity, complete O2 binding curves at acid pH could be obtained only using a high pressure (up to 24 atm) optical cell with pure O2 [40]. The Hill plot of trout HbIV displays heme–heme interactions (nH >> 1) at pH around 8–7 but at more acidic pH the ligand affinity drops dramatically and the curve becomes initially flat and eventually clearly biphasic. The pH 6.1 data were interpreted [27] within a model based on (a) non-cooperative O2 binding to the T-state and (b) very different affinities of the two types of subunits a and b (see the values of nH = 1 below and above Y ~ 0.5). Assigning the two equilibrium phases at pH 6.1 to the a and b subunits is consistent with optical spectroscopy [41] and high-resolution NMR of 13 CO, which clearly discriminates the tertiary structure of the a and b subunits [42]. In trout HbIV, the affinity of the low affinity subunit at pH 6.1 is indeed very low, p1/2 being well above 2 atm of pure O2 (and even at 20 atm only ~ 95% saturation was achieved) [40]. The evolutionary significance of this peculiar behavior raised the curiosity of physiologists and biochemists alike. The presence of 638
such an extreme pH effect in the blood of teleost fish has been correlated with the function of the swim bladder, the organ that allows teleost fish to maintain neutral buoyancy at any depth [43]. Under extreme hydrostatic pressures in deep-water fishes (say around 100 atm), pumping O2 into the swim bladder is hard, but can be achieved because of (a) the production of lactic acid by the gas gland (an organ associated with the swim bladder); (b) the rete mirabile, a network of blood vessels engineered to maximize small molecule exchange by countercurrent flow; and (c) the presence of an Hb that rapidly senses the drop in pH and allosterically activates a mechanism to drastically reduce O2 affinity. The pH shift experienced at the level of the gas gland induces dissociation of O2 because of an allosteric conformational change that triggers the onset of extremely low affinity (Table 2). From examination of Fig. 3C, the overall O2 affinity shift in trout HbIV amounts to > 1000-fold from pH 8 to pH 6, a dramatic event of molecular adaptation to physiological requirements. A few comments concerning the interpretation of this extreme allosteric control are in order. This large heterotropic effect (classically called the Root effect [39]) is linked to protonation of selected key residue(s) that not only shift the quaternary T-to-R equilibrium stabilizing the former state but also affect the tertiary structure of the heme cavity, predominantly in one of the two types of subunit, thereby lowering O2 affinity even further. The effect of low pH in stabilizing the T-state of fully liganded Hb has been demonstrated by a variety of methods. For example, in the case of trout HbIV fully saturated with CO, a spectral shift in the Soret band discovered by Giardina et al. [41] was found consistent with the stabilization of T4 and with spectral analysis of T3↔R3 obtained using photochemical modulation [44,45]. Among other experimental data, the most informative and definitive stem from solving the 3D structure of tuna fish Hb (a typical Root effect Hb) by X-ray crystallography, a difficult task and a success due to Tame and his collaborators [46]. The molecular mechanism to explain the stabilization of the T-state by acidic pH is still debated. One hypothesis [47] was based on Perutz’s stereochemical mechanism [7] whereby the T-state of HbA is stabilized by salt bridges between the imidazole of the C-terminal His146b and Asp or Glu94 or Glu90 of the same subunit (which raises its pK), and between the C-terminal carboxyl of the same His146b and Lys40a. In mammals, the SH group of Cys93b forms no H-bonds with its neighbors and takes up one of two alternative positions in the R-state, while in the T-state FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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Cys93b is out of the Tyr pocket but screened from solvent by His146b. In fish Hbs displaying a Root effect, Cys93b is replaced by Ser whose side chain in the Tstate is engaged in H-bonds with His146b and with the peptide NH of the same His, stabilizing the C-terminal salt bridges and increasing the allosteric constant, decreasing in O2 affinity and raising the pKa of His146b. This hypothesis, however, was sharply criticized by Yokoyama et al. [46] despite some data on carp Hb which appeared consistent; these authors pointed out, however, that substitution of Cys93b by Ser in HbA did not produce the Root effect (reported in [46]) and that, in the T-state tuna Hb, Asp94b makes an H-bond with Ser93b but is too far from His146b. Based on the crystallographic structure of other fish Hbs [48,49], alternative hypotheses have been proposed including a destabilization of the R-state; however, a final convincing explanation is yet to come. In the Root effect Hbs, the most interesting peculiarity is the large inequality in O2 binding between the two types of subunits in the T-state, so crucial to achieve an extreme decrease of affinity (Fig. 3C). Two questions are in order: (a) is it the a or the b subunit that acquires the extremely low O2 affinity (with p1/2 at ~ 2–4 atm of pure O2) and (b) what is the underlying pH dependent molecular mechanism? The crystallographic data of Yokoyama et al. [46] on tuna Hb, obtained for the unliganded T-state at pH 5 and 7.5 and the CO-bound R-state at pH 8, revealed some interesting pH-linked conformational changes unique to fish. A novel T-state intra-chain salt bridge between His69b and Asp72b breaks upon CO binding, a tertiary interaction presumed to stabilize T and reduce O2 affinity. Moreover in the T-state a proton is shared between Asp96a1 and Asp101b2 both interacting with Ser98a by H-bonding; this interaction, which is broken in going to R, is across the a1b2 interface and thus directly relevant to the allosteric switch. A peculiar and intriguing conformational change involving distal His60a (topological position E7) was identified [46] in the structure of one of the two a subunits in the deoxy T-state, in going from neutral to acid pH (Fig. 4). At pH 7.5 His46a is canonically facing the heme iron in the binding pocket and most probably contributes by H-bonding to the stability of bound O2 [12,25]. However, at pH 5 this distal His60a swings away from the cavity and makes an alternative H-bond with one of the two propionates of the heme (replacing His46a that makes this H-bond at pH 7.5); a concerted motion involving Trp47a is also detected, as shown in Fig. 4. This set of conformational changes triggered (most likely) by the protonation of the FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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Fig. 4. The heme pocket of the a subunit of tuna fish deoxy Hb. The 3D structure of the heme pocket of the a2 subunit of tuna fish Hb obtained by crystallography on deoxygenated crystals at pH 7.5 (in red, PDB 1V4W) or pH 5.0 (in blue, PDB 1V4X) (data by Yokoyama et al. [46]). The heme is shown edge-on and the E-helix with the distal His(E7)60a is above; the proximal His(F8)89a remains put at acid pH. It may be noticed that at pH 5 there is a concerted motion of the imidazole of His(CE3)46a and of Trp(CE4) 47a moving outside, while His(E7)60a shifts away and makes a bond (salt bridge) with propionate 1 of the heme. Polar bonds shown as broken lines in red (pH 7.5) or blue (pH 5.0).
imidazole of His60a is expected to be associated with the loss of stabilizing interactions between bound O2 and the distal His, and thereby with a considerable drop in O2 affinity. In fact it has been demonstrated by mutagenesis and transient spectroscopy ([50–52] and references therein) that the O2 dissociation rate constant increases up to more than 1000-fold upon substitution of the distal His60a with Gly or other side chains. This may well be the crucial structural change accounting for the very low affinity seen at acid pH for the a subunits and responsible for the peculiar biphasic O2 binding curve typical of teleost fish Hbs (shown in Fig. 3C). In summary, the most likely hypothesis for the very low affinity that is engineered to pump O2 into the swim bladder is a property of the a subunits. The pH-linked conformational change involving distal His60a and coupled motions of Trp47a and His46a is evidence for the tertiary allosteric control mechanism underlying this effect.
Destabilizing the T-state for high O2 affinity Among the striking variants discovered by investigating respiration physiology of different vertebrates, the case of birds flying at high altitudes [53] proved particularly intriguing. A paradigmatic case is the barheaded goose (Anser indicus), a bird that lives and hatches at high altitudes in Tibet (~ 4000–6000 m) and 639
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when migrating to the plains flies across and above Mount Everest breathing air at pO2 of approximately one-third the value at sea level (see [54] and references therein). This remarkable survival skill of maintaining sufficient O2 reserve to support strenuous physical activity even at extremely high altitudes is associated with modifications of the Hb molecule that lead to a destabilization of the T-state and thereby an increase in ligand affinity, sufficient to capture O2 even at very low pressures. The significant mutations were identified by comparing the sequences of the a and b subunits of the bar-headed goose with those of the low-land living relatives, i.e. the greylag goose, the Canada goose and the mute swan [54]. Only four mutations were identified, and by reasonable considerations it was concluded that a significant mutation typical of birds
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and likely to affect function is Pro119a?Ala. This Pro is located at the a1b1 interface edge (Fig. 5A) [55–57], touching Leu55b, a contact that is impossible with the shorter Ala mutant. It is fascinating indeed that a parallel evolutionary adaptation has been discovered in the Andean goose (Chloephaga melanoptera) living in South America at altitudes of 5000–6000 m [53,58]; similarly to what is reported for the bar-headed goose, its Hb is also endowed with higher O2 affinity compared with low-land relatives. In the latter case, one replacement seemed particularly significant among all those identified in the a and b subunits [59], namely the mutation Leu55b?Ser, facing Pro119a across the a1b1 interface; such a mutation would introduce a gap in the contact area. The molecular mechanism whereby this specific replacement is associated with a considerable increase
A
B
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Fig. 5. High affinity bird Hbs. (A) Structure of oxygenated Hb from the bar-headed goose, obtained by crystallography (from [57] with permission of JRH Tame). Electron density map of the a1b1 contact region, contributed by Ala(H2)119a and Leu(D6)55b; the position of the ring of Pro (H2)119a present in wild-type human HbA is superimposed, highlighted as a broken line. (B) Hill plots of the O2 binding equilibria of normal human HbA (open circles, to the right) and of the single site directed mutant Pro(H2)119a ?Ala (open squares, to the left), synthesized to mimic the high O2 affinity of the bar-headed goose Hb (see text). Experimental conditions: pH 7.3, < 5 mMCl and 25 °C. The higher overall affinity of the mutant is correlated to an increase in the O2 affinity of the T-state (KT), as shown by the position of the bottom asymptote of the curve on the left (from [60], with permission of RE Weber).
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in O2 affinity has been investigated in some detail. Already Perutz [16] suggested that a limited but significant perturbation of the a1b1 interface would be associated with a destabilization of the T-state, by deleting the contact between Pro119a and Met55b1 in HbA (Fig. 5A); this would lead to an increase in O2 affinity without abolishing heme–heme interactions (that are nevertheless reduced). The 3D structure of the barheaded goose oxy Hb [57] showed the overall structure to be similar to that of human HbO2 but the Pro119a?Ala mutation leads to loss of direct interaction with residue Leu55b across the interface (the side chain of Ala119a being much too short). It is astonishing that two species of high flying birds (namely the bar-headed goose and the Andean goose) have evolved two different replacements in the very same a1b1 contact region that, without disrupting the interface, are sufficient to destabilize the T-state and therefore shift the energy level diagram by reducing L0. It should be appreciated that mutating human HbA by introducing either of the two substitutions (i.e. Pro119a?Ala and Met55b?Ser) led to completely consistent results (see Fig. 5B): higher O2 affinity of both mutants and somewhat reduced cooperativity (p1/2 ~ 3.3 mmHg and nH ~ 1.5–2.0 at pH 7.2, 25 °C, and 0.1 MCl , compared with p1/ 2 ~ 5.8 mmHg and nH ~ 3 for HbA) [54,60]. This effect of destabilizing the T-state by perturbing the a1b1 interface should correspond to ~ 1.0–1.5 kcal per tetramer. It may be recalled that in human HbA Amiconi et al. [61] modified Met55b by oxidation to the sulfoxide changing the polarity of the side chain drastically, with a dramatic effect on function: in fact O2 binding was found to be non-cooperative (nH = 1) and high affinity (similar to that of the R-state), implying a destabilization of the T-state definitely more dramatic than that exploited by natural selection with mutation Met55b?Ser.
ulous resurrection of interest, to the genuine satisfaction of Changeux (personal communication). Indeed, under the pressure of evidence for functionally linked conformational changes experienced by more and more proteins solved by X-ray diffraction or by NMR, the application of the allosteric control model has extended considerably encompassing complex oligomeric systems (up to the ribosome) down to intriguing monomeric proteins, such as some natively unfolded transcription factors [65]. An appealing and professional review that I would recommend has been published by Cui and Karplus [66]. Some people ask why the MWC allosteric model based on conformational selection has enjoyed such an enormous success for so many years. In my opinion several factors have contributed: the blunt recognition of a crucial role for pre-existing conformational states of the protein was the conceptual step; the model’s predictive power as well as its formal elegance and beauty; the intuition and the courage to introduce the term allosteric, a striking but unexpected success of classical education; and the substantial adherence to selection which is the accepted mechanism for survival of the fittest. After all, Dobzhansky [67] stated that ‘Nothing in Biology makes sense except in the light of Evolution’; and as written by Henri Buc [5], ‘the «MWC» article bears Monod’s label, an amateur in physico-chemistry, a visionary in so far as molecular Darwinism is concerned’.
Acknowledgements I would like to express my deep appreciation to Dr.ssa Stefania Contardi for invaluable help in the setting of this manuscript, to Prof.ssa Adriana E. Miele for helpful suggestions and for the preparation of Fig. 4, and to Dr. Giorgio Giardina for the preparation of Fig. 3.
References Epilogue From the late 1960s, Jean-Pierre Changeux fell in love with the nicotinic acetylcholine receptor and his experiments successfully extended the basic idea of allosteric control to functional membranes [62]. His work has had a considerable impact not only in biochemistry and molecular biology but also in neurophysiology and pharmacology, and the concept of allosteric modulator is currently familiar in the field of ligand-gated ion channels and G-coupled receptors ([2,63,64] and references therein). It is a fact that after a period of fairly limited popularity the MWC allosteric theory experienced a miracFEBS Journal 281 (2014) 633–643 ª 2013 FEBS
1 Changeux J-P (2011) 50th anniversary of the word ‘allosteric’. Protein Sci 20, 1119–1124. 2 Changeux J-P (2013) The origins of allostery: from personal memories to material for the future. J Mol Biol 425, 1396–1406. 3 Monod J, Changeux J-P & Jacob F (1963) Allosteric proteins and cellular control systems. J Mol Biol 6, 306–329. 4 Monod J, Wyman J & Changeux J-P (1965) On the nature of allosteric transitions: a plausible model. J Mol Biol 12, 88–118. 5 Buc H (2006) Interactions between Jacques Monod and Jeffries Wyman (or the Burdens of co-Authorship). In:
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40 Brunori M, Coletta M, Giardina B & Wyman J (1978) A macromolecular transducer as illustrated by trout hemoglobin IV. Proc Natl Acad Sci USA 75, 4310–4312. 41 Giardina B, Ascoli F & Brunori M (1975) Spectral changes and allosteric transition in trout haemoglobin. Nature 256, 761–762. 42 Giacometti GM, Giardina B, Brunori M, Giacometti G & Rigatti G (1976) Observations on CO trout hemoglobins by 13C NMR. FEBS Lett 62, 157–160. 43 Denton EJ (1961) The buoyancy of fish and cephalophods. Prog Biophys Biophys Chem 11, 177–234. 44 Martino AJ & Ferrone FA (1989) Rate of allosteric change in hemoglobin measured by modulated excitation using fluorescence detection. Biophys J 56, 781–794. 45 Bellelli A & Brunori M (1994) Optical measurements of quaternary structural changes in hemoglobin. Methods Enzymol 232, 56–71. 46 Yokoyama T, Chong KT, Miyazaki G, Morimoto H, Shih DT, Unzai S, Tame JR & Park SY (2004) Novel mechanisms of pH sensitivity in tuna hemoglobin: a structural explanation of the Root effect. J Biol Chem 279, 28632–28640. 47 Perutz MF & Brunori M (1982) Stereochemistry of cooperative effects in fish and amphibian haemoglobins. Nature 299, 421–426. 48 Camardella L, Caruso C, D’Avino R, di Prisco G, Rutigliano B, Tamburrini M, Fermi G & Perutz MF (1992) Haemoglobin of the antarctic fish Pagothenia bernacchii. Amino acid sequence, oxygen equilibria and crystal structure of its carbonmonoxy derivative. J Mol Biol 224, 449–460. 49 Milvaganam SE, Bonaventura C, Bonaventura J & Getzoff ED (1996) Structural basis for the Root effect in haemoglobin. Nat Struct Biol 3, 275–283. 50 Nagai K, Luisi B, Shih D, Miyazaki G, Imai K, Poyart C, De Young A, Kwiatkowsky L, Noble RW, Lin SH et al. (1987) Distal residues in the oxygen binding site of haemoglobin studied by protein engineering. Nature 329, 858–860. 51 Olson JS, Mathews AJ, Rohlfs RJ, Springer BA, Egeberg KD, Sligar SG, Tame J, Renaud JP & Nagai K (1988) The role of the distal histidine in myoglobin and haemoglobin. Nature 336, 265–266. 52 Birukou I, Schweers RL & Olson JS (2010) Distal histidine stabilizes bound oxygen and acts as a gate for ligand entry in both subunits of adult human hemoglobin. J Biol Chem 285, 8840–8854. 53 Hall FG, Dill DB & Guzman-Barron ES (1936) Comparative physiology in high altitudes. J Cell Comp Physiol 8, 301–313. 54 Jessen TH, Weber RE, Fermi G, Tame J & Braunitzer G (1991) Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by
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Variations on the theme: allosteric control in hemoglobin Maurizio Brunori Istituto Pasteur – Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Italy
Keywords allosteric models; evolution of hemoglobin; heterotropic effects; physiological requirements; structure and function Correspondence M. Brunori, Dipartimento di Scienze Biochimiche, Sapienza University of Rome, P. le Aldo Moro 5, 00185 Rome, Italy Fax: +39 06 49910717 Tel: +39 06 4450291 E-mail: [email protected]
Conformational selection between pre-existing structural states of an oligomeric protein was the conceptual step behind the formulation of Monod– Wyman–Changeux (MWC) allosteric theory. Variations on the basic theme of allosteric control are briefly illustrated in this paper by reference to some hemoglobins from different species whose functional properties were found to respond to specific physiological requirements. In my opinion the enormous success of the allosteric theory may be attributed not only to its efficiency in accounting for data and its formal mathematical elegance, but also because the selective mechanism conforms to the founding concept of Darwinian evolution.
(Received 22 July 2013, revised 10 September 2013, accepted 23 September 2013) doi:10.1111/febs.12586
Forward According to the records, the term ‘allosteric inhibition’ was coined in 1961 by Jacques Monod and Francßois Jacob in a written comment to the paper presented by Jean-Pierre Changeux at the Cold Spring Harbor Symposium on Quantitative Biology; the history of the events that led to the fundamental findings and the conception of the theoretical model have been presented by Changeux himself ([1,2] and references therein). After the first full paper that appeared in 1963 [3], the theory was elaborated and formalized in the paper by Monod, Wyman and Changeux [4] published in the Journal of Molecular Biology in May 1965. This seminal work proved to be most inspiring, a remarkable success for a theoretical paper that over the years has been extensively cited. An interesting historical account of the scientific interactions between Jacques Monod and Jeffries Wyman (from January 1963 to January 1965) and the chronology of the events was presented by Henri Buc [5] at a meeting commemorating the 40th
anniversary of the Monod–Wyman–Changeux (MWC) model, held at the Accademia Nazionale dei Lincei in Rome in May 2005. It is not difficult to understand why hemoglobin (Hb) became the paradigmatic protein to illustrate the theory of allosteric control. In the early 1960s a great deal of innovative quantitative information was already available and carefully analyzed: the 3D structure of Hb had been solved by Max Perutz et al. [6,7] and shown to be different in the oxy and deoxy states; cooperative O2 binding and the Bohr effect had been extensively studied [8,9]; several natural mutants of human Hb (including some pathological variants) and the Hbs from many different species of vertebrates had been investigated [10]; and much more … [11,12]. In 1951 a seminal idea had been conceived by Wyman and Allen [13] as a result of an analysis of quantitative data on the Bohr effect, much before any knowledge of the structure of the protein became available.
Abbreviations BPG, 2,3-bisphosphoglycerate; Hb, hemoglobin.
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Interpreting the Bohr effect, Wyman and Allen wrote: ‘the reason why certain acid groups are affected by oxygenation is simply the alteration of their position and environment which results from the change of configuration of the hemoglobin molecule as a whole accompanying oxygenation, …’. It was realized that proteins with their complexity of possible configurations (nowadays called the ‘conformational landscape’) would be fitted to play the role of an enzyme, a prophetic idea that years later justified the promotion of Hb to the rank of an honorary enzyme: ‘if we are prepared to accept hemoglobin as an enzyme, its behavior might give us a hint as to the kind of process to be looked for in enzymes more generally’ [13]. In 1966 Koshland, Nemethy and Filmer [14] published an alternative model based on the concept of induced fit, whereby binding of a substrate induces a conformational change in the relevant subunit of the oligomeric enzyme. Thus, contrary to the basic concept of allostery, induced fit is a sequential model with population of the reaction intermediate states. The KNF model has been extensively applied especially in molecular enzymology, being originally conceived to account for enzyme specificity. It would demand a whole paper to highlight the relative merits of the MWC and KNF models in describing the behavior of Hb; and clearly this is not the place. Still it seemed only fair to recall the seminal contribution of Dan Koshland and collaborators [14,15], generally illustrated in all biochemistry textbooks. In this short account I shall not deal in any detail with the paradigmatic case of human HbA, probably the most extensively studied protein, but will highlight the general consistency with the allosteric model of the peculiar extreme behavior of some interesting Hbs from fish and birds.
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emerged as the common answer in vertebrates. All of them synthesize a2b2 tetrameric Hb as the canonical O2 carrier, characterized by allosteric conformational control to maximize efficiency in delivery. Allostery is the general mechanism adopted in the evolution of vertebrates to provide all cells in the body with sufficient O2 for energy production. The large variability in O2 demands under very different environmental conditions (changes in temperature, pressure, pH, O2 concentration etc.) has been confronted with peculiar molecular responses [16] adapting the efficiency of O2 delivery by Hb to respond to specific and variable physiological demands. Particularly interesting and challenging has been the correlation between the physiological needs in the context of a specific environment and the modifications of the structural and functional properties of Hb brought about by mutation and selection [16], maintaining the mechanism of allosteric control to optimize O2 delivery. In Wyman’s words [17]: ‘The theme is hemoglobin; the variations, the differences in its properties and behavior which have been developed in the course of evolution to meet special requirements of different animal forms’. Extending the study of physiology and biochemistry of blood to many species relies on nature doing the work of chemical modification. The scientific expedition to the Amazon in 1976 was justified by the curiosity to investigate the exotic species of fish living in that mother of rivers. This paper written for the jubilee of the allosteric theory highlights a few paradigmatic examples of fascinating molecular adaptation to peculiar physiological requirements as observed in different animal species, and their consistency with the basic features of the MWC theory.
The energy level diagram The demand for an oxygen carrier A dramatic revolution in biological evolution has been the transition from a reductive to an oxidizing atmosphere with a large increase in the partial pressure of O2 produced by photosynthesis. The opportunity for more efficient energy production, that eventually culminated in the mitochondrial oxidative phosphorylation, made the difference. The emergence of vertebrates and the general increase in body mass demanded huge energy production/ATP synthesis and thereby some appropriate devices to make O2 available to every cell of every organ in the body, given that gas diffusion proved inadequate for large multicellular organisms. Circulation of blood and the synthesis of a super-specialized carrier cell, the erythrocyte, have 634
The essential framework of the allosteric theory is depicted in Fig. 1A in terms of a diagram indicating the energy levels of the two quaternary allosteric states of a tetramer with four chemically identical subunits, each binding a ligand [18,19]. In going from top to bottom, the scheme highlights the increasing number of ligands bound from zero (deoxyHb) to four (fully oxygenated Hb). In the absence of ligand, the population ratio between states is expressed by the fundamental constant L0 = [T0]/[R0], which for HbA may vary between 103 and 107 (depending on experimental conditions). The a and b subunits in tetrameric a2b2Hb are characterized by subtle differences in tertiary structure that may play a crucial role in functional control. FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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A
B
Fig. 1. Energetics of the MWC model, and the Hill plot. (A) Energy level diagram indicating the two different quaternary states (squares and circles) and the 10 ligation species (T0 to T4 and R0 to R4). The fundamental equilibrium constants of the MWC model [4] are shown: KT and KR, the oxygen dissociation constants of the two allosteric states T (tense) and R (relaxed); and L0, the population ratio of the two states in the fully deoxygenated tetrameric Hb. The implicit assumption is that for a fully concerted quaternary transition the switch-over point will be at the level T2– R2 in the case of a symmetric binding curve. Typical values of the parameters for human HbA at neutral pH and 20 °C are ~ L0 = [T0]/ [R0] = 105 and c = KR/KT = 0.01. The allosteric equilibrium constant depends on the number of ligands bound, according to the equation Li = L0ci, with i from 0 to 4. (B) Hill plot of the oxygen equilibrium of horse Hb in 0.6 M phosphate buffer at pH 7.0 and 19 °C; different techniques were employed to acquire data in the different saturation ranges going from < 1% to ~ 99%. Total free energy of interaction DFi = 2.6 kcalmol 1 (heme); nH = 2.95. Data of RLJ Lyster as reported by Wyman [9], modified.
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The basic components of the model are (a) the presence of two structural states of the oligomer (R and T) that are populated at every level of saturation; and (b) the difference in ligand affinity between the two allosteric states, defined by the dissociation equilibrium constants KR and KT [4]. In the scheme of Fig. 1A, T0 is more populated than R0 and thus, despite its lower O2 affinity (KR 1000-fold, then binding may occur to the R-state albeit the least populated of species, and the Hill coefficient will tend to nH = 1. In addition, since all molecular species are always present at equilibrium, if L0 decreases considerably due (for example) to destabilization of the T-state, the binding will tend to occur preferentially by the R-state and thus O2 affinity will be high (approaching that of KR) and cooperativity low; and vice versa when the T-state is heavily stabilized, with a large increase in L0. These reciprocal relationships were analyzed in a formal paper by Wyman [21] and then elaborated by several other authors [12,18,22]. Figure 2 depicts the early analysis by Stuart Edelstein [22] who presented some of the experimental data available on HbA and some natural mutants, to highlight the correlation. Among the many examples arising from studies on human HbA under different conditions, a beautiful and particularly informative set of data on HbA trapped in crystals or in silica gel has been published by Mozzarelli et al. [23]. Trapping deoxyHb (in the T0-state) stabilizes a species that binds O2 with no apparent cooperativity and very low affinity, which has been interpreted as a single molecular state with characteristic tertiary and quaternary structures. This model has been extended to kinetics and is the basis for a novel attractive mechanism for human HbA, championed by Eaton et al. [24]. 635
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Fig. 2. Correlation between the Hill coefficient and the allosteric equilibrium constant. A classical demonstration of the dependence of the Hill coefficient nH (estimated between Y = 0.25 and Y = 0.75) on log L0 yielding a bell shaped curve, as predicted by the MWC model (see text). Numbers indicate data for HbA at two pH values; other data are for the isolated a and b subunits; (C) human Hb Chesapeake (Leu(FG4)92a?Arg); (S) stripped human HbA; (K) human Hb Kansas (Asn (G4)102b?Tyr) (from [22], with permission of SJ Edelstein).
The minimalist allosteric hemoglobin Figure 3 depicts the O2 binding isotherms of different Hbs that vary widely in their overall affinity and dependence of shape on pH and/or solvent composition. Human adult HbA (Fig. 3B) has been very extensively investigated [7,11,12,18,20,25] and is obviously the reference system throughout the field. The peculiar example depicted in Fig. 3A represents the O2 binding equilibrium of trout HbI, one of the components expressed in the blood of trout [26,27]. As may be seen from examination of the Hill plot, trout HbI is clearly cooperative with thermodynamic parameters [28] that are fairly similar to those of HbA under canonical experimental conditions, the maximum Hill coefficient being nH = 2.0–2.2 compared with nH = 2.8–3.0 for HbA and the overall free energy of interaction being (heme) compared with DFi = 2.2 kcalmol 1 DFi = 3.0 kcalmol 1 (heme) for HbA (see the distance between asymptotes). Trout HbI was shown to populate two different structural states based on a number of physical properties, the most direct being the 3D structure of the deoxy and CO derivatives solved by resolution. We have Tame et al. [29] at 2.2–2.4 A recently analyzed in depth the structural constraints and weak bonds that break in going from trout HbI deoxy (T0) to the CO bound state (R4) [30]. We concluded that the salt bridges broken during the allosteric transition [7,31] make a significant contribution to 636
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the difference in stability between the allosteric states, but cannot account alone for the overall free energy change calculated from the O2 binding isotherm. Several features of the structure–function relationships have been highlighted by Miele et al. [30]. The extraordinary feature of trout HbI is that the shape and position of the O2 binding curve is totally independent of solvent composition: no Bohr effect, no 2,3-bisphosphoglycerate (BPG) (or other phosphates) effect and indeed a very limited (almost zero) effect of temperature on p(O2)1/2 [26,27]. Moreover there is no evidence for ab subunit inequivalence; no significant dissociation into ab dimers even below micromolar concentration; and a switch-over point just above 2 ([30] and references therein). The dynamics of trout HbI have been extensively studied using time resolved laser spectroscopy [32] carried out over a wide range of fractional photolysis and temperatures from 2 to 65 °C. Results of this analysis compared with equilibrium data [28,33,34] confirmed the switch-over point to be close to doubly liganded, and the relaxation time for the R0?T0 transition around 20 ls, close to that determined for HbA by Sawicki and Gibson [35]. The lack of heterotropic effects (pH and phosphates) is quite peculiar. It was understood based on the findings obtained by amino acid sequence analysis [36]; as shown in Table 1, the residues that Perutz [7,25] proposed to be involved in the Bohr effect and in the organic phosphates effect are either absent or substituted by ineffective amino acids. This represents fully independent evidence in support of the classical assignment, and indeed almost proof for the mechanism of differential binding of protons and phosphate linked to oxygenation. In summary, trout HbI functional behavior is fully consistent with a ‘pure’ idealized MWC model, as depicted in Fig. 1A. However, the ligand-linked conformational equilibrium between R and T (Table 2) is totally insensitive to solvent composition, and thus efficiency of O2 delivery is solely based on homotropic interactions. The only variable affecting (somewhat) the relative population of states is temperature, with a standard enthalpy change for the T0R0 transition of ~ 30 kcalmol 1 of tetramer [28,34]. Since the population of the R0 state increases with temperature, L0 was calculated to be 1 at ~ 75–80 °C and naturally the Hill coefficient would tend to nH = 1. This prediction was confirmed by flash photolysis experiments on trout HbI-CO carried out up to ~ 72 °C [37]. Such a minimalist allosteric Hb responds effectively to tissue O2 demand but it is of limited latitude. It was proposed [27] that this type of Hb was selected for a FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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A
B
Fig. 3. Oxygen equilibrium curves of human and trout Hbs. Oxygen binding data are plotted in terms of a Hill plot, where Y is the fractional saturation with the ligand and pO2 is the oxygen partial pressure in the gas phase. (A) O2 binding curves of trout HbI at two pH values (indicated), in 0.2 M Bistris buffer at 4 °C (from [28], redrawn). (B) O2 binding curves of human HbA at different pH values (indicated) and 20 °C. In all cases 0.1 MCl was present; the curve on the right at pH 7.4 (triangles) contains also 2 mM inositol-hexaphosphate (from [20], redrawn). (C) O2 binding curves of trout HbIV at four different pH values (indicated), in 0.05 M Bistris buffer at 14 °C; a high pressure optical cell was employed for the very low affinity range (from [40], redrawn).
C
specific vital purpose expressed in fast swimming teleost fishes, while sluggish fish like carp do not express this type of Hb. In the case of hyperactive fish living in fast running waters, an Hb devoid of heterotropic control and representing a significant fraction (35%– 40%) of the total Hb in the blood fulfills the role of an emergency O2 supplier under critical metabolic demands related to strenuous exercise. In fact in hyperactive fish the production of lactic acid may be FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
sufficient to produce significant blood acidity which inhibits O2 supply to the tissues; the animal may suffer from internal asphyxia. The case of catostomid fish studied by Dennis Powers [38] is a particularly attractive example, given that members belonging to the subgenus Catostomus that live in fast moving waters express trout HbI-like carriers, whereas members of another subgenus (Pantosteus) living preferentially in pools do not. 637
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Table 1. Comparison between human HbA and trout HbI.
a a b b b b
subunit subunit subunit subunit subunit subunit
Human HbA
Trout HbI
Heterotropic ligand
Val1 (NH1) His122 (H5) His2 (NH2) Lys82 (EF6) His143 (H21) His146 (HC3)
Ac-Ser1 His123 Glu2 Leu82 Ser143 Phe146
Proton (Bohr effect) Proton (Bohr effect) BPG BPG BPG Proton (Bohr effect)
Amino acid substitutions directly linked to heterotropic allosteric effects; the topological positions are in parentheses.
Table 2. Allosteric equilibrium constant L0 for human and trout hemoglobins, at different pH values and 23–25 °C. L0 ([T0]/[R0]) pH 7.4 Trout HbI Human HbA Trout HbIV
3
3 x 10 8.7 x 104 4 x 103
pH 6.5 3
3 x 10 6.2 x 105 1 x 105
pH 6.0 3 x 103 3 x 106
Data from [20] and [28].
Extreme heterotropic control The O2 binding curves depicted in Fig. 3C are representative of the peculiar functional behavior common to all teleost fish Hbs, as reported long ago by Root [39]. In the case of trout, approximately 60% of the total Hb is characterized by such an extreme pH dependence of the shape and overall affinity [26]; given the very low affinity, complete O2 binding curves at acid pH could be obtained only using a high pressure (up to 24 atm) optical cell with pure O2 [40]. The Hill plot of trout HbIV displays heme–heme interactions (nH >> 1) at pH around 8–7 but at more acidic pH the ligand affinity drops dramatically and the curve becomes initially flat and eventually clearly biphasic. The pH 6.1 data were interpreted [27] within a model based on (a) non-cooperative O2 binding to the T-state and (b) very different affinities of the two types of subunits a and b (see the values of nH = 1 below and above Y ~ 0.5). Assigning the two equilibrium phases at pH 6.1 to the a and b subunits is consistent with optical spectroscopy [41] and high-resolution NMR of 13 CO, which clearly discriminates the tertiary structure of the a and b subunits [42]. In trout HbIV, the affinity of the low affinity subunit at pH 6.1 is indeed very low, p1/2 being well above 2 atm of pure O2 (and even at 20 atm only ~ 95% saturation was achieved) [40]. The evolutionary significance of this peculiar behavior raised the curiosity of physiologists and biochemists alike. The presence of 638
such an extreme pH effect in the blood of teleost fish has been correlated with the function of the swim bladder, the organ that allows teleost fish to maintain neutral buoyancy at any depth [43]. Under extreme hydrostatic pressures in deep-water fishes (say around 100 atm), pumping O2 into the swim bladder is hard, but can be achieved because of (a) the production of lactic acid by the gas gland (an organ associated with the swim bladder); (b) the rete mirabile, a network of blood vessels engineered to maximize small molecule exchange by countercurrent flow; and (c) the presence of an Hb that rapidly senses the drop in pH and allosterically activates a mechanism to drastically reduce O2 affinity. The pH shift experienced at the level of the gas gland induces dissociation of O2 because of an allosteric conformational change that triggers the onset of extremely low affinity (Table 2). From examination of Fig. 3C, the overall O2 affinity shift in trout HbIV amounts to > 1000-fold from pH 8 to pH 6, a dramatic event of molecular adaptation to physiological requirements. A few comments concerning the interpretation of this extreme allosteric control are in order. This large heterotropic effect (classically called the Root effect [39]) is linked to protonation of selected key residue(s) that not only shift the quaternary T-to-R equilibrium stabilizing the former state but also affect the tertiary structure of the heme cavity, predominantly in one of the two types of subunit, thereby lowering O2 affinity even further. The effect of low pH in stabilizing the T-state of fully liganded Hb has been demonstrated by a variety of methods. For example, in the case of trout HbIV fully saturated with CO, a spectral shift in the Soret band discovered by Giardina et al. [41] was found consistent with the stabilization of T4 and with spectral analysis of T3↔R3 obtained using photochemical modulation [44,45]. Among other experimental data, the most informative and definitive stem from solving the 3D structure of tuna fish Hb (a typical Root effect Hb) by X-ray crystallography, a difficult task and a success due to Tame and his collaborators [46]. The molecular mechanism to explain the stabilization of the T-state by acidic pH is still debated. One hypothesis [47] was based on Perutz’s stereochemical mechanism [7] whereby the T-state of HbA is stabilized by salt bridges between the imidazole of the C-terminal His146b and Asp or Glu94 or Glu90 of the same subunit (which raises its pK), and between the C-terminal carboxyl of the same His146b and Lys40a. In mammals, the SH group of Cys93b forms no H-bonds with its neighbors and takes up one of two alternative positions in the R-state, while in the T-state FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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Cys93b is out of the Tyr pocket but screened from solvent by His146b. In fish Hbs displaying a Root effect, Cys93b is replaced by Ser whose side chain in the Tstate is engaged in H-bonds with His146b and with the peptide NH of the same His, stabilizing the C-terminal salt bridges and increasing the allosteric constant, decreasing in O2 affinity and raising the pKa of His146b. This hypothesis, however, was sharply criticized by Yokoyama et al. [46] despite some data on carp Hb which appeared consistent; these authors pointed out, however, that substitution of Cys93b by Ser in HbA did not produce the Root effect (reported in [46]) and that, in the T-state tuna Hb, Asp94b makes an H-bond with Ser93b but is too far from His146b. Based on the crystallographic structure of other fish Hbs [48,49], alternative hypotheses have been proposed including a destabilization of the R-state; however, a final convincing explanation is yet to come. In the Root effect Hbs, the most interesting peculiarity is the large inequality in O2 binding between the two types of subunits in the T-state, so crucial to achieve an extreme decrease of affinity (Fig. 3C). Two questions are in order: (a) is it the a or the b subunit that acquires the extremely low O2 affinity (with p1/2 at ~ 2–4 atm of pure O2) and (b) what is the underlying pH dependent molecular mechanism? The crystallographic data of Yokoyama et al. [46] on tuna Hb, obtained for the unliganded T-state at pH 5 and 7.5 and the CO-bound R-state at pH 8, revealed some interesting pH-linked conformational changes unique to fish. A novel T-state intra-chain salt bridge between His69b and Asp72b breaks upon CO binding, a tertiary interaction presumed to stabilize T and reduce O2 affinity. Moreover in the T-state a proton is shared between Asp96a1 and Asp101b2 both interacting with Ser98a by H-bonding; this interaction, which is broken in going to R, is across the a1b2 interface and thus directly relevant to the allosteric switch. A peculiar and intriguing conformational change involving distal His60a (topological position E7) was identified [46] in the structure of one of the two a subunits in the deoxy T-state, in going from neutral to acid pH (Fig. 4). At pH 7.5 His46a is canonically facing the heme iron in the binding pocket and most probably contributes by H-bonding to the stability of bound O2 [12,25]. However, at pH 5 this distal His60a swings away from the cavity and makes an alternative H-bond with one of the two propionates of the heme (replacing His46a that makes this H-bond at pH 7.5); a concerted motion involving Trp47a is also detected, as shown in Fig. 4. This set of conformational changes triggered (most likely) by the protonation of the FEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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Fig. 4. The heme pocket of the a subunit of tuna fish deoxy Hb. The 3D structure of the heme pocket of the a2 subunit of tuna fish Hb obtained by crystallography on deoxygenated crystals at pH 7.5 (in red, PDB 1V4W) or pH 5.0 (in blue, PDB 1V4X) (data by Yokoyama et al. [46]). The heme is shown edge-on and the E-helix with the distal His(E7)60a is above; the proximal His(F8)89a remains put at acid pH. It may be noticed that at pH 5 there is a concerted motion of the imidazole of His(CE3)46a and of Trp(CE4) 47a moving outside, while His(E7)60a shifts away and makes a bond (salt bridge) with propionate 1 of the heme. Polar bonds shown as broken lines in red (pH 7.5) or blue (pH 5.0).
imidazole of His60a is expected to be associated with the loss of stabilizing interactions between bound O2 and the distal His, and thereby with a considerable drop in O2 affinity. In fact it has been demonstrated by mutagenesis and transient spectroscopy ([50–52] and references therein) that the O2 dissociation rate constant increases up to more than 1000-fold upon substitution of the distal His60a with Gly or other side chains. This may well be the crucial structural change accounting for the very low affinity seen at acid pH for the a subunits and responsible for the peculiar biphasic O2 binding curve typical of teleost fish Hbs (shown in Fig. 3C). In summary, the most likely hypothesis for the very low affinity that is engineered to pump O2 into the swim bladder is a property of the a subunits. The pH-linked conformational change involving distal His60a and coupled motions of Trp47a and His46a is evidence for the tertiary allosteric control mechanism underlying this effect.
Destabilizing the T-state for high O2 affinity Among the striking variants discovered by investigating respiration physiology of different vertebrates, the case of birds flying at high altitudes [53] proved particularly intriguing. A paradigmatic case is the barheaded goose (Anser indicus), a bird that lives and hatches at high altitudes in Tibet (~ 4000–6000 m) and 639
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when migrating to the plains flies across and above Mount Everest breathing air at pO2 of approximately one-third the value at sea level (see [54] and references therein). This remarkable survival skill of maintaining sufficient O2 reserve to support strenuous physical activity even at extremely high altitudes is associated with modifications of the Hb molecule that lead to a destabilization of the T-state and thereby an increase in ligand affinity, sufficient to capture O2 even at very low pressures. The significant mutations were identified by comparing the sequences of the a and b subunits of the bar-headed goose with those of the low-land living relatives, i.e. the greylag goose, the Canada goose and the mute swan [54]. Only four mutations were identified, and by reasonable considerations it was concluded that a significant mutation typical of birds
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and likely to affect function is Pro119a?Ala. This Pro is located at the a1b1 interface edge (Fig. 5A) [55–57], touching Leu55b, a contact that is impossible with the shorter Ala mutant. It is fascinating indeed that a parallel evolutionary adaptation has been discovered in the Andean goose (Chloephaga melanoptera) living in South America at altitudes of 5000–6000 m [53,58]; similarly to what is reported for the bar-headed goose, its Hb is also endowed with higher O2 affinity compared with low-land relatives. In the latter case, one replacement seemed particularly significant among all those identified in the a and b subunits [59], namely the mutation Leu55b?Ser, facing Pro119a across the a1b1 interface; such a mutation would introduce a gap in the contact area. The molecular mechanism whereby this specific replacement is associated with a considerable increase
A
B
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Fig. 5. High affinity bird Hbs. (A) Structure of oxygenated Hb from the bar-headed goose, obtained by crystallography (from [57] with permission of JRH Tame). Electron density map of the a1b1 contact region, contributed by Ala(H2)119a and Leu(D6)55b; the position of the ring of Pro (H2)119a present in wild-type human HbA is superimposed, highlighted as a broken line. (B) Hill plots of the O2 binding equilibria of normal human HbA (open circles, to the right) and of the single site directed mutant Pro(H2)119a ?Ala (open squares, to the left), synthesized to mimic the high O2 affinity of the bar-headed goose Hb (see text). Experimental conditions: pH 7.3, < 5 mMCl and 25 °C. The higher overall affinity of the mutant is correlated to an increase in the O2 affinity of the T-state (KT), as shown by the position of the bottom asymptote of the curve on the left (from [60], with permission of RE Weber).
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in O2 affinity has been investigated in some detail. Already Perutz [16] suggested that a limited but significant perturbation of the a1b1 interface would be associated with a destabilization of the T-state, by deleting the contact between Pro119a and Met55b1 in HbA (Fig. 5A); this would lead to an increase in O2 affinity without abolishing heme–heme interactions (that are nevertheless reduced). The 3D structure of the barheaded goose oxy Hb [57] showed the overall structure to be similar to that of human HbO2 but the Pro119a?Ala mutation leads to loss of direct interaction with residue Leu55b across the interface (the side chain of Ala119a being much too short). It is astonishing that two species of high flying birds (namely the bar-headed goose and the Andean goose) have evolved two different replacements in the very same a1b1 contact region that, without disrupting the interface, are sufficient to destabilize the T-state and therefore shift the energy level diagram by reducing L0. It should be appreciated that mutating human HbA by introducing either of the two substitutions (i.e. Pro119a?Ala and Met55b?Ser) led to completely consistent results (see Fig. 5B): higher O2 affinity of both mutants and somewhat reduced cooperativity (p1/2 ~ 3.3 mmHg and nH ~ 1.5–2.0 at pH 7.2, 25 °C, and 0.1 MCl , compared with p1/ 2 ~ 5.8 mmHg and nH ~ 3 for HbA) [54,60]. This effect of destabilizing the T-state by perturbing the a1b1 interface should correspond to ~ 1.0–1.5 kcal per tetramer. It may be recalled that in human HbA Amiconi et al. [61] modified Met55b by oxidation to the sulfoxide changing the polarity of the side chain drastically, with a dramatic effect on function: in fact O2 binding was found to be non-cooperative (nH = 1) and high affinity (similar to that of the R-state), implying a destabilization of the T-state definitely more dramatic than that exploited by natural selection with mutation Met55b?Ser.
ulous resurrection of interest, to the genuine satisfaction of Changeux (personal communication). Indeed, under the pressure of evidence for functionally linked conformational changes experienced by more and more proteins solved by X-ray diffraction or by NMR, the application of the allosteric control model has extended considerably encompassing complex oligomeric systems (up to the ribosome) down to intriguing monomeric proteins, such as some natively unfolded transcription factors [65]. An appealing and professional review that I would recommend has been published by Cui and Karplus [66]. Some people ask why the MWC allosteric model based on conformational selection has enjoyed such an enormous success for so many years. In my opinion several factors have contributed: the blunt recognition of a crucial role for pre-existing conformational states of the protein was the conceptual step; the model’s predictive power as well as its formal elegance and beauty; the intuition and the courage to introduce the term allosteric, a striking but unexpected success of classical education; and the substantial adherence to selection which is the accepted mechanism for survival of the fittest. After all, Dobzhansky [67] stated that ‘Nothing in Biology makes sense except in the light of Evolution’; and as written by Henri Buc [5], ‘the «MWC» article bears Monod’s label, an amateur in physico-chemistry, a visionary in so far as molecular Darwinism is concerned’.
Acknowledgements I would like to express my deep appreciation to Dr.ssa Stefania Contardi for invaluable help in the setting of this manuscript, to Prof.ssa Adriana E. Miele for helpful suggestions and for the preparation of Fig. 4, and to Dr. Giorgio Giardina for the preparation of Fig. 3.
References Epilogue From the late 1960s, Jean-Pierre Changeux fell in love with the nicotinic acetylcholine receptor and his experiments successfully extended the basic idea of allosteric control to functional membranes [62]. His work has had a considerable impact not only in biochemistry and molecular biology but also in neurophysiology and pharmacology, and the concept of allosteric modulator is currently familiar in the field of ligand-gated ion channels and G-coupled receptors ([2,63,64] and references therein). It is a fact that after a period of fairly limited popularity the MWC allosteric theory experienced a miracFEBS Journal 281 (2014) 633–643 ª 2013 FEBS
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