Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

Daugherty, Margaret A.; Shea, Madeline A.; Johnson, Jeremy A.; LiCata, Vince J.; Turner, George J.; Ackers, Gary K.

doi:10.1073/pnas.88.4.1110

Cited by 44 publications

(65 citation statements)

References 27 publications

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“…7), indicating that Asymmetric effect of single desArg modification at the first ligand binding step. The first CNmet ligand, denoted as X, binds to either ␣-(shown) or ␤-subunit in wild-type Hb (gray subunits) with a ⌬G c penalty of 3.0 Ϯ 0.2 kcal͞mol at pH 7.4 and 21.5°C (38). When both ␣141Arg residues are removed (green subunits), effectively eliminating ␣ 1 -␣ 2 contacts between the two dimers, the penalty is reduced to 1.0 Ϯ 0.2 kcal͞mol.…”

Section: Resultsmentioning

confidence: 99%

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

Ackers

Dalessio

Lew

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism of cooperativity in the human hemoglobin tetramer (a dimer of ␣␤ dimers) has historically been modeled as a simple two-state system in which a low-affinity structural form (T) switches, on ligation, to a high-affinity form (R), yielding a net loss of hydrogen bonds and salt bridges in the dimer-dimer interface. Modifications that weaken these cross-dimer contacts destabilize the quaternary T tetramer, leading to decreased cooperativity and enhanced ligand affinity, as demonstrated in many studies on symmetric double modifications, i.e., a residue site modified in both ␣-or both ␤-subunits. In this work, hybrid tetramers have been prepared with only one modified residue, yielding molecules composed of a wild-type dimer and a modified dimer. It is observed that the cooperative free energy of ligation to the modified dimer is perturbed to the same extent whether in the hybrid tetramer or in the doubly modified tetramer. The cooperative free energy of ligation to the wild-type dimer is unperturbed, even in the hybrid tetramer, and despite the overall destabilization of the T tetramer by the modification. This asymmetric response by the two dimers within the same tetramer shows that loss of dimer-dimer contacts is not communicated across the dimer-dimer interface, but is transmitted through the dimer that bears the modified residue. These observations are interpreted in terms of a previously proposed dimer-based model of cooperativity with an additional quaternary (T͞R) component.M any tertiary and quaternary conformational events important for catalysis and cooperativity in biological macromolecules are energetically unfavorable, and must be driven by favorable free energy of ligand binding. In the case of human hemoglobin (Hb), initial binding of O 2 to the heme Fe drives intrinsically unfavorable conformation change within the globin. The associated energetic ''penalty'' results in lower binding constants for initial ligation. However, penalties for the additional binding steps are progressively reduced, generating Hb's characteristic sigmoidal binding curve. Contributing to this curve are the partially ligated Hb intermediates, composed of two different subunit types (␣ and ␤) with four binding sites (␣ 1 , ␤ 1 , ␣ 2 , ␤ 2 ). Up to four labile ligands (O 2 ) are present on each tetramer in multiple configurations of site occupancy within at least two distinct quaternary structures (T and R). The challenge in understanding the mechanism of Hb cooperativity has been to identify and measure the separate energetic penalties for each O 2 binding step, and then to correlate individual penalties with specific structural events.To approach a resolution of this complex problem, energetic components of the system's ligation intermediates were evaluated with no prior assumptions as to their quaternary structures. Analysis of the thermodynamic linkages between dimertetramer assembly and O 2 binding permitted the ligand binding constant of the noncooperative free dimer to be used as the thermodynamic reference s...

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Section: Resultsmentioning

confidence: 99%

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

Ackers

Dalessio

Lew

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…For the Fe/Fe + data, the calculated ratio of oxygen affinities is 237 (ZZG of 3.2 kcal/mol); this value was apparently rounded off to 3 kcal/mol when reported as a factor of 170 [3,94]. Application of this definition (pathway 01-11-21) does not seem to have been uniformly employed; the origin of the reported value of 40 for the Fe/Fe-O 2 (equivalent to the Zn/Fe) is not obvious; Huang et al [34] indicated an energy of 2.2 kcalmol (factor of 40), but this value is only the ZG between 11 and 21 tetramers. It was pointed out (by N. Shibayama) that the correct value based on the reported free energies is [(14.4− 11.6)− (11.6−9.4)]= 0.6 kcal/mol or a factor of 2.8, which is consistent with the simple two-state model.…”

Section: Discussionmentioning

confidence: 99%

Identifying the conformational state of bi-liganded haemoglobin

Marden

Kiger

Poyart

et al. 1998

Cellular and Molecular Life Sciences (CMLS)

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While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992). Science 255: 54-63]. The implications of this model and the relevant experiments will be reviewed here.

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“…The original evidence for this preferred ligation pathway was the hyper (170-fold) stability of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) relative to the other three doubly liganded species (7)(8)(9). However, this evidence has been proven to be an artifact arising from valency exchange between the deoxy and cyanometheme sites during the long deoxy incubation that had been used routinely in the laboratory of Ackers (11,12).…”

Section: O 2 Equilibrium Properties Of [␣(Fe)␤(fe)][␣(zn)␤(zn)]-di-mentioning

confidence: 99%

“…a 170-fold affinity change). Subsequently, the effects of pH, temperature, and single-site mutations on the dimer-tetramer equilibrium of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) were studied (7,9,10). It was suggested that this key intermediate assumes a form of the deoxy-Tquaternary structure as judged from the nature of the dimerdimer interface.…”

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confidence: 99%

“…Based on the hyperthermodynamic stability and T quaternary structure of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤), Ackers' group proposed a new framework for Hb cooperativity, called a symmetry rule (SR), in 1991 (9). The key features of the SR model are: (i) The two ligation steps leading to the asymmetric ␣1␤1 half-liganded intermediate show distinctly favorable cooperativity while those leading to the other three doubly liganded intermediates exhibit no favorable cooperativity; (ii) The asymmetric ␣1␤1 half-liganded intermediate assumes a deoxy-T-quaternary structure while the other three doubly liganded intermediates exhibit an oxy-R-quaternary structure.…”

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confidence: 99%

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The Contribution of the Asymmetric α1β1 Half-oxygenated Intermediate to Human Hemoglobin Cooperativity

Yun

Morimoto

Shibayama

2002

Journal of Biological Chemistry

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Although cooperative oxygenation of human adult hemoglobin (HbA) 1 has been studied extensively as a paradigm for regulatory actions of allosteric proteins, the functional and structural properties of its eight partially oxygenated intermediates remain elusive. This is mostly due to the strong cooperativity of Hb, which suppresses relative abundance of the intermediates, precluding direct study. One of the most specific methods for studying the oxygenation intermediates has been to substitute the heme in one or more of four subunits with another metalloprotoporphyrin IX, which does not bind O 2 while mimicking either normal deoxyheme or oxyheme (1-6). However, there has been a limitation to this approach: Six asymmetric forms cannot be studied in isolation, because the asymmetric hybrid tetramers dissociate into dimers, which then reassociate to form not only the former asymmetric tetramers but also symmetric ones (i.e. dimer rearrangement).Ackers and his colleagues (7) have partly circumvented this difficulty by measuring the dimer-tetramer equilibrium constants for asymmetric hybrid species in the presence of the symmetric parental species. In 1985, this methodology was first applied to resolution of the tetramer stabilities of all ten ligation intermediates of the deoxy-cyanomet [Fe(II)/ Fe(III)-CN Ϫ ] hybrid system, in which cyanometheme mimics a fixed oxyheme (8). The most striking finding was that the dimer-tetramer association equilibrium constant for (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) was about 170 times that of the other three doubly liganded species at pH 7.4, which implies a hypercooperativity in specific ligation steps in the ␣1␤1 dimer within the tetramer (i.e. a 170-fold affinity change). Subsequently, the effects of pH, temperature, and single-site mutations on the dimer-tetramer equilibrium of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) were studied (7, 9, 10). It was suggested that this key intermediate assumes a form of the deoxy-Tquaternary structure as judged from the nature of the dimerdimer interface.Based on the hyperthermodynamic stability and T quaternary structure of (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤), Ackers' group proposed a new framework for Hb cooperativity, called a symmetry rule (SR), in 1991 (9). The key features of the SR model are: (i) The two ligation steps leading to the asymmetric ␣1␤1 half-liganded intermediate show distinctly favorable cooperativity while those leading to the other three doubly liganded intermediates exhibit no favorable cooperativity; (ii) The asymmetric ␣1␤1 half-liganded intermediate assumes a deoxy-T-quaternary structure while the other three doubly liganded intermediates exhibit an oxy-R-quaternary structure.Recently, we showed that both the published hyperstability and T structure assignment for (␣ ϩCNϪ ␤ ϩCNϪ )(␣␤) were artifacts arising from valency exchange between the deoxy and cyanometheme sites during the long deoxy incubation that had been used routinely in the laboratory of Ackers (11,12). Ackers et al. (13) have now accepted the valency exchange artifacts in their previous studies using de...

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Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

Cited by 44 publications

References 27 publications

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

Single residue modification of only one dimer within the hemoglobin tetramer reveals autonomous dimer function

Identifying the conformational state of bi-liganded haemoglobin

The Contribution of the Asymmetric α1β1 Half-oxygenated Intermediate to Human Hemoglobin Cooperativity

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