Heterotropic Allosteric Effectors Research Articles

Dissimilar flexibility of α and β subunits of human adult hemoglobin influences the protein dynamics and its alteration induced by allosteric effectors.

The general question by what mechanism an “effector” molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability.

Ion-regulated allosteric binding of fullerenes (C60 and C70) by tetrathiafulvalene-calix[4]pyrroles.

The effect of ionic species on the binding of fullerenes (C60 and C70) by tetrathiafulvalene-calix[4]pyrrole (TTF-C4P) receptors and the nature of the resulting supramolecular complexes (TTF-C4P + fullerene + halide anion + tetraalkylammonium cation) was studied in the solid state through single crystal X-ray diffraction methods and in dichloromethane solution by means of continuous variation plots and UV-vis spectroscopic titrations. These analyses revealed a 1:1 stoichiometry between the anion-bound TTF-C4Ps and the complexed fullerenes. The latter guests are bound within the bowl-like cup of the C4P in a ball-and-socket binding mode. The interactions between the TTF-C4P receptors and the fullerene guests are highly influenced by both the nature of halide anions and their counter tetraalkylammonium cations. Three halides (F(-), Cl(-), and Br(-)) were studied. All three potentiate the binding of the two test fullerenes by inducing a conformational change from the 1,3-alternate to the cone conformer of the TTF-C4Ps, thus acting as positive heterotropic allosteric effectors. For a particular halide anion, the choice of tetraalkylammonium salts serves to modulate the strength of the TTF-C4P-fullerene host-guest binding interactions and, in conjunction with variations in the halide anion, can be exploited to alter the inherent selectivity of the host for a given fullerene. Differences in binding are reflected in the excited state optical properties. Overall, the present four-component system provides an illustration of how host-guest binding events involving appropriately designed artificial receptors can be fine-tuned via the addition of simple ionic species as allosteric modulators.

Allosterically Tunable, DNA-Based Switches Triggered by Heavy Metals

Here we demonstrate the rational design of allosterically controllable, metal-ion-triggered molecular switches. Specifically, we designed DNA sequences that adopt two low energy conformations, one of which does not bind to the target ion and the other of which contains mismatch sites serving as specific recognition elements for mercury(II) or silver(I) ions. Both switches contain multiple metal binding sites and thus exhibit homotropic allosteric (cooperative) responses. As heterotropic allosteric effectors we employ single-stranded DNA sequences that either stabilize or destabilize the nonbinding state, enabling dynamic range tuning over several orders of magnitude. The ability to rationally introduce these effects into target-responsive switches could be of value in improving the functionality of DNA-based nanomachines.

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O 2-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O 2-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O 2-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O 2-affinities ( K T = 0.0085 mmHg −1 and K R = 0.011 mmHg −1) and thus a very low cooperativity ( K R / K T = 1.3 and L 0 = 2.4). 1H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O 2-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, 1H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14 ppm, originated from inter- dimeric α 1β 2- (or α 2β 1-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15–25 ppm, caused by high-spin heme-Fe(II)s. Upon addition of O 2, the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O 2, but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O 2 ( P O2 ) of 760 mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P O2 of 760 mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α 1G- and β 1G-helices, and the other weaker binding site causes the R → T transition without release of O 2. The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α 1β 1 (or α 2β 2) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α 1α 2- (or β 1β 2-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O 2-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb — [ the deoxy-state] = [ the T-quaternary structure] = [ the low O 2 -affinity state] and [ the oxy-state] = [ the R-quaternary structure] = [ the high O 2 -affinity state] and the O 2 -affiny of Hb being regulated by the T/R-quaternary structural transition — are no longer sustainable . This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Thermodynamic Profiles of Heterotropic Allostery of Hemoglobin (Hb) by Isothermal Titration Calorimetry (ITC)

The O2-affinities of deoxy- and oxy-states of Hb (KTand KR) are reduced up to 60- and 2,000-folds, respectively, by interactions with potent heterotropic allosteric effectors such as BPG, IHP, BZF, and L35 (1,2). These heterotropic allosteries of such magnitudes mean that heterotropic allosteries are the principal regulatory function of Hb rather than the homotropic allostery (cooperativity), since the latter provides an allostery of only ∼30-folds (1). It should be noted that such hetrotropic allosteries are accompanied by no static quaternary or tertiary structural changes (3). Even α- and β-semi-Hb, though they are αβ-dimers, exhibit a heterotropic allostery of up to 100-folds (4). Thermodynamic profiles of these heterotropic allosteries of Hb have been measured by ITC, in order to assess the nature of the interactions of Hb with heterotropic allosteric effectors. Supported by an NIH grant, HL14508.

Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: Inhibition of E. coli glycerol kinase

Unlike those for monomeric superfamily members, heterotropic allosteric effectors of the tetrameric Escherichia coli glycerol kinase (EGK) bind to only one of the two domains that define the catalytic cleft and far from the active site. An R369A amino acid substitution removes oligomeric interactions of a novel mini domain-swap loop of one subunit with the catalytic site of another subunit, and an A65T substitution perturbs oligomeric interactions in a second interface. Linked-functions enzyme kinetics, analytical ultracentrifugation, and FRET are used to assess effects of these substitutions on the allosteric control of catalysis. Inhibition by phosphotransferase system protein IIA Glc is reduced by the R369A substitution, and inhibition by fructose 1,6-bisphosphate is abolished by the A65T substitution. The oligomeric interactions enable the heterotropic allosteric effectors to act on both domains and modulate the catalytic cleft closure despite binding to only one domain.

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl-, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (alphaFeO2)2-(betaZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70-200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl-. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.

Allostery in chaperonins

Chaperonins mediate protein folding in an ATP-dependent manner. ATP binding and hydrolysis by chaperonins are subject to both homotropic and heterotropic allosteric regulation. In the case of GroEL and CCT, homotropic regulation by ATP is manifested in nested cooperativity, which involves positive intra-ring cooperativity and negative inter-ring cooperativity in ATP binding. Both types of cooperativity are modulated by various heterotropic allosteric effectors, which include nonfolded proteins, ADP, Mg2+, monovalent ions such as K+, and cochaperonins in the case of type I chaperonins such as GroEL. Here, the allosteric properties of chaperonins are reviewed and new results of ours are presented with regard to allosteric effects of ADP. The role of allostery in the reaction cycle and folding function of chaperonins is discussed.

High pressure reveals that the stability of interdimeric contacts in the R- and T-state of HbA is influenced by allosteric effectors: Insights from computational simulations

The molecular details of the mechanism of action of allosteric effectors on hemoglobin oxygen affinity are not clearly understood. The global allostery model proposed by Yonetani et al. suggests that the binding of allosteric effectors can take place both in the R and T states and that they influence oxygen affinity through inducing global tertiary changes in the subunits. Recently published high pressure studies yielded dissociation constants at atmospheric pressure that showed a stabilizing effect of heterotropic allosteric effectors on the dimer interface in the R state, and a more pronounced destabilizing effect in a T state model. In the present work, we report on computational modeling used to interpret the high pressure experimental data. We show structural changes in the hemoglobin interdimeric interfaces, indicative of a global tertiary structural change induced by the binding of allosteric effectors. We also show that the number of water molecules bound at the interface is significantly influenced by binding effectors in the T state in accordance with the experimental data. Our results suggest that the binding of effectors at definite sites leads to tertiary changes that propagate to the interfaces and results in overall structural re-organizations.

Allosteric modulation of monomeric proteins*

Multimeric proteins (e.g. hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric proteins (e.g. myoglobin) usually are assumed to be nonallosteric. However, the modulation of the functional properties of monomeric proteins by heterotropic allosteric effectors casts doubts on this assumption. Here, the allosteric properties of sperm whale myoglobin, human serum albumin, and human α-thrombin, generally considered as molecular models of monomeric proteins, are summarized.

Semihemoglobins, High Oxygen Affinity Dimeric Forms of Human Hemoglobin Respond Efficiently to Allosteric Effectors without Forming Tetramers

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated.

Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA.

Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.

The global allostery model of hemoglobin: an allosteric mechanism involving homotropic and heterotropic interactions

Studies of the allosteric mechanism of hemoglobin (Hb) have evolved from phenomenological descriptions to structure-based molecular mechanisms, as the molecular structures of Hb in deoxy and ligated states have been elucidated. The MWC two-state concerted model has been the widely accepted as the most plausible of the allosteric mechanisms of Hb. It assumes that the O 2-affinity of Hb is regulated/controlled primarily by the T/R quaternary structural transition and that heterotropic effectors bind preferentially to T (deoxy) Hb to shift the T/R allosteric equilibrium toward the T state. However, recent more comprehensive O 2-binding measurements of Hb have revealed a new mechanism, the Global Allostery model. It describes that the O 2-affinity and the cooperativity are modulated in greater extents and the Bohr effect is generated primarily by the tertiary structural changes in both T (deoxy) and R (ligated) states of Hb. Differential interactions of heterotropic allosteric effectors with both T (deoxy) and R (ligated) states of Hb induce these tertiary structural changes. The X-ray structure of a complex of R (ligated) Hb with BZF, a potent heterotropic effector, has revealed the stereo-chemical influence of these effectors on the structure of R (ligated) Hb, resulting in the reduction of the ligand affinity in R (ligated) Hb. This model stresses the fundamental importance of the heterotropic interactions in regulation/control of the functionality of Hb. They alter the tertiary structures of both T (deoxy) and R (oxy) Hb, leading to large-scale modulations of the O 2 affinity ( K T and K R), and consequently the cooperativity ( K R/ K T) and the Bohr effect (Δ P 50/ΔpH) from a global viewpoint of allostery in Hb. To cite this article: T. Yonetani, A. Tsuneshige, C. R. Biologies 326 (2003).

Global Allostery Model of Hemoglobin: MODULATION OF O2 AFFINITY, COOPERATIVITY, AND BOHR EFFECT BY HETEROTROPIC ALLOSTERIC EFFECTORS

The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin.

The pathway of allosteric control as revealed by hemoglobin intermediate states

The energetics of hemoglobin cooperativity has been analyzed through the use of stable, partially-ligated intermediates. These studies revealed that the two dimeric halves of the tetramer are autonomous, leading to a Symmetry Rule that governs the relationship between ligand-binding and the T-->R quaternary switch: the R structure is favored over T only when ligands are bound to both dimers within the tetramer. A major feature of the Symmetry Rule mechanism is the generation of cooperative free energy by tertiary conformational constraints, which are formed within one dimeric half of the T-tetramer and released during the quaternary structure change to R. These rules of tertiary and quaternary molecular switching also govern the roles of the heterotropic allosteric effectors (e.g. Bohr protons).

Aspartate Transcarbamylase: A STUDY OF POSSIBLE ROLES FOR THE SULFHYDRYL GROUP AT THE ACTIVE SITE

Abstract Potential roles for the single sulfhydryl group of the catalytic subunit of aspartate transcarbamylase from Escherichia coli have been investigated by means of chemical modification. The S-cyano and S-methyl derivatives are highly active and recombine normally with the zinc regulatory subunit. The derivatives of native enzyme formed in this way are fully responsive to homotropic and heterotropic allosteric effectors and have pH-activity curves indistinguishable from that of unmodified native enzyme. Therefore, despite strong evidence that the—SH group is at the active site, its integrity is not necessary for activity or for allosteric function. However, modifications which lead to negatively charged derivatives of the—SH group do result in inactivation. The reason for such loss of activity was investigated with a sulfonate derivative prepared by rapid and highly specific oxidation of the native enzyme with KMnO4. Carbamyl phosphate and the transition state analog N-(phosphonacetyl)-l-aspartate bind well to the sulfonate derivative, but the affinity of this derivative for succinate (in the presence of carbamyl-P) is much reduced when compared to that of native enzyme. The newly introduced negative charge apparently inactivates by greatly reducing the affinity of the active site for the substrate l-aspartate. The trimeric catalytic subunit is inactive after modification of the 3 identical —SH groups by dithionitrobenzoate, and the derivative is capable of binding tightly only 1 eq of either carbamyl-P or N-(phosphonacetyl)-l-aspartate per trimer, demonstrating a strong interaction among the three active sites of the isolated subunit. Furthermore, the thionitrobenzoate derivative of native aspartate transcarbamylase correspondingly binds only about 2 molecules of N-(phosphonacetyl)-l-aspartate per six active sites and binds them with strong positive cooperativity, suggesting that in unmodified native enzyme positive cooperativity is due at least in part to an interaction between the two catalytic trimers. If catalytic subunit is combined with a limiting amount of zinc regulatory subunit, a new species deficient in regulatory subunit is formed, with altered allosteric properties.

Factors affecting the oxidation of glycerol-1-phosphate by insect flight-muscle mitochondria

The oxidation of l-glycerol-1-phosphate by flight-muscle mitochondria isolated from a variety of insects has been studied. Low levels of calcium ions, 10 −7 M, and strontium ions, 10 −5 M, act as heterotropic allosteric effectors of the oxidation by both mitochondria and sub-mitochondrial particles. l-Glycerol-1-phosphate also acts as a homotropic effector in the absence of calcium and strontium ions. The preparation is described of submitochondrial particles, EGTA particles, which do not show allosteric behaviour. The site of action of calcium and strontium ions is discussed.