Confirmation of a unique intra-dimer cooperativity in the human hemoglobin ?1?1half-oxygenated intermediate supports the symmetry rule model of allosteric regulation

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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...

Section: Methodsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

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

Ackers

Dalessio

Lew

et al. 2002

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“…To put the cooperativity in perspective, human I-BABP can be compared with human hemoglobin. The largest cooperativity factor in hemoglobin, which occurs with the binding of the fourth molecule of oxygen, is Ϸ400 (14). For glycocholate binding to human I-BABP, the cooperativity factor exceeds 1,000.…”

Section: Discussionmentioning

confidence: 98%

Energetics by NMR: Site-specific binding in a positively cooperative system

Tochtrop

Richter

Tang

et al. 2002

146

Proteins with multiple binding sites exhibit a complex behavior that depends on the intrinsic affinities for each site and the energetic communication between the sites. The contributions from intrinsic affinity and cooperativity are difficult to deconvolute using conventional binding experiments that lack information about the occupancies of individual sites. Here, we report the concerted use of NMR and isothermal titration calorimetry to determine the intrinsic and cooperative binding free energies for a ligand-protein complex. The NMR measurements provided the site-specific information necessary to resolve the binding parameters. Using this approach, we observed that human ileal bile acid binding protein binds two molecules of glycocholic acid with low intrinsic affinity but an extraordinarily high degree of positive cooperativity. The highly cooperative nature of the binding provides insights into the protein's biological mechanism. With ongoing improvements in sensitivity and resolution, NMR methods are becoming more amenable to dissecting the complex binding energetics of multisite systems.

“…A series of recent findings, however, suggest that, despite this structural equivalence, the ␣ 1 ␤ 1 and ␣ 2 ␤ 2 interfaces are not functionally inert. Ackers et al have demonstrated that, when the second ligand binds to Hb, the microscopic species in which both ligands are on the same ␣␤ dimer is energetically preferred over all other doubly ligated species (8,35). This result implies the existence of favorable cooperativity between ␣ and ␤ hemes within the same ␣␤ dimer, before the T 3 R transition.…”

Section: Allosteric Free Energy Changes At His␣103(g10) and His␣122(hmentioning

confidence: 99%

A signature of the T → R transition in human hemoglobin

Mihailescu

Russu

2001

Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are His␣103(G10) and His␣122(H5) in each ␣ subunit of hemoglobin. They are located at the ␣1␤1 and ␣2␤2 interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the ␤ chains. We have measured the solvent exchange rates of side chain protons of His␣103(G10) and His␣122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by Ϸ1.5 kcal͞(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric ␣1␤1 and ␣2␤2 interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.T he Hb molecule has been a paradigm for understanding the molecular events underlying biological function. Cooperative oxygen-binding and allosteric effects have been rationalized by global thermodynamic models that postulate states of the molecule with specific energetic features (1-3). In the two-state model proposed by Monod, Wyman, and Changeux (2), the two energetic states are the tense (T) and the relaxed (R) states. The two states differ from each other in their affinity for ligand and in the interactions between subunits. Binding of oxygen to the deoxygenated (deoxy)-T state occurs with low affinity. However, the binding of the ligand destabilizes the T state and makes it more likely to switch to the high-affinity oxygenated (oxy)-R state. Thus, as ligation proceeds, the gradual enrichment in Hb molecules in the R state accounts for cooperativity in ligand binding. The success of this model in explaining the function of Hb has promoted its general application to a large variety of systems, including allosteric protein systems and nucleic acidbinding proteins (reviewed in refs. 4 and 5). However, the structural basis of this thermodynamic model is not yet fully understood.The first insights into the molecular structures representing the T and R thermodynamic states of Hb have been provided by crystallographic results on deoxy and ligated Hb obtained by Perutz (6, 7). The deoxy-T and ligated-R structures maintain the same ␣ 1 ␤ 1 and ␣ 2 ␤ 2 interfaces within ␣␤ dimers but differ greatly at the ␣ 1 ␤ 2 and ␣ 2 ␤ 1 interfaces between the two d...