Mutagenic dissection of hemoglobin cooperativity: Effects of amino acidalteration on subunit assembly of oxy and deoxy tetramers

Turner, George J.; Galactéros, Frédéric; Doyle, Michael L.; Hedlund, Bo E.; Pettigrew, Donald W.; Turner, Benjamin W.; Smith, F. B.; Moo-Penn, Winston F.; Rucknagel, Donald L.; Ackers, Gary K.

doi:10.1002/prot.340140303

Cited by 80 publications

(111 citation statements)

References 57 publications

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“…3). In contrast, the double mutant of Hb Bunbury exhibits tetramer stability and ligand affinity indistinguishable from that of normal HbA 0 (29), whereas the double mutant of Hb Yakima destabilizes the deoxy tetramer by 4.5 kcal͞mol and stabilizes the fully ligated tetramer by 1.9 kcal͞mol, comparable to that found with the O 2 ligand (29). The total cumulative ⌬G c penalty is 6.2 kcal͞mol for double Bunbury and only 0.1 kcal͞mol for double Yakima, compared with 6.0 kcal͞mol for wild type.…”

Section: Resultsmentioning

confidence: 81%

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: 81%

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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“…In hemoglobin, the T (unbound)-to-R (bound) transition occurs when one O 2 molecule binds to the heme group, causing a conformational change that displaces the F helix, inducing a shift of the intersubunit contacts that stabilize the high-affinity O 2 state (Turner et al 1992;Royer et al 2001). The binding of a ligand at one site that causes subsequent conformational changes that affect ligand binding at another site is a fundamental mechanism inclusive of other allosteric proteins (Copeland, 2000) and in the design of allosteric ribozymes (Koizumi et al 1999;Jose et al 2001).…”

Section: A Mechanism For Cooperative Glycine Bindingmentioning

confidence: 99%

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Erion¹,

Strobel²

2010

RNA

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The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.

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“…Human HbA undergoes dissociation to dimers more readily in its oxygenated compared to its deoxygenated form (Nagel & Gibson, 1967;Benesch et al, 1976; Turner et al, 1992). Like oxy HbA, the D99K(P) mutant Hb in its oxygenated state dissociated readily to form dimers that combined with haptoglobin (Table 3).…”

Section: Dissociation Of the Mutant Hbmentioning

confidence: 99%

Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

Yanase¹,

Cahill²,

Llano³

et al. 1994

Protein Science

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Site-directed mutagenesis of an important subunit contact site, , by a Lys residue (D99K(@)) was proven by sequencing the entire @-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(@) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the a-and @-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(@) recombinant mutant forms have differences in their heme-protein environments.

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Mutagenic dissection of hemoglobin cooperativity: Effects of amino acidalteration on subunit assembly of oxy and deoxy tetramers

Cited by 80 publications

References 57 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

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

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