Binding Of Inositol Hexaphosphate Research Articles

Modulation of hemoglobin dynamics by an allosteric effector.

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.

Solution Structure and Dynamics of Human Hemoglobin in the Carbonmonoxy Form

The solution structure of human adult carbonmonoxy hemoglobin (HbCO A) was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous nuclear magnetic resonance structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution at low salt concentrations resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A. Amide-water proton exchange experiments demonstrated that αThr38 located in the α1β2 interface and several loop regions in both α- and β-chains were dynamic on the subsecond time scale. Side chain methyl dynamics revealed that methyl groups in the α1β2 interface were dynamic, but those in the α1β1 interface were quite rigid on the nanosecond to picosecond and millisecond to microsecond time scales. All the data strongly suggest a dynamic α1β2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamic changes in the α1β2 interface and the regions around the hemes but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the millisecond time scale, very likely arising from the relative motion of the α1β1 dimer with respect to the α2β2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Determination of Oxygen Binding Properties of the Individual Subunits of Intact Human Adult Hemoglobin

Abstract We determined the oxygen (O2) binding properties of the individual subunits of intact human adult hemoglobin (Hb A) through the combined use of 1H NMR and conventional spectrophotometric methods. We found that the O2 affinities of the α and β subunits of Hb A are almost indistinguishable in the absence of an allosteric effector, inositol hexaphosphate (IHP), and lower by factors of ≈8.4 and ≈10, respectively, in the presence of IHP at pH 7.40, as judged from the estimated P50 values, which represent the partial O2 pressure required to achieve 50% oxygenation of the individual subunits. These results demonstrated that the binding of IHP to the protein resulted in remarkable enhancement of the degree of nonequivalence in functional properties between the constituent subunits, which could be relevant to its cooperative ligand binding. This finding provides novel insights for elucidation of the molecular mechanisms responsible for the cooperativity in this allosteric protein.

Equilibrium Reaction Of Human Methemoglobin A

A study has been carried out on the sulfhydryl group reactivities of human methemoglobin A and its azide complex with 2,2\'-dithiobispyridine (2-DTP) in the presence and absence of inositol hexaphosphate (IHP) in order to evaluate electrostatic effects on reactivity and the relationship between conformational states and reactivity. The reaction was monitored spectrophotometrically by the formation of 2-thiopyridone (the product of the reaction) with an absorption peak at 343 nm at 20 °C. The binding of IHP to methemoglobin A (met-HbA) diminishes the reactivity of β-93 sulfhydryl group towards 2DTP. This effect was used to determine the pH profile of the binding constants of IHP to the surface of the protein while changing the complex at the heme iron. Work was also done on the equilibrium reaction of azide ion with IHP- and 2DTP- chemically modified met-Hb A as a function of pH, and an equilibrium relationship was developed for reactions both at the heme iron and at the surface of the methemoglobin molecule. The result shows that binding at the heme by ligands such as azide ion affects the orientation of charge groups on the surface of the met-Hb molecule as well as binding of organic phosphate. IFE Journal of Science Vol. 9 (2) 2007 pp. 225-230

Effector-induced structural fluctuation regulates the ligand affinity of an allosteric protein: binding of inositol hexaphosphate has distinct dynamic consequences for the T and R states of hemoglobin.

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.

Insights into the Solution Structure of Human Deoxyhemoglobin in the Absence and Presence of an Allosteric Effector

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.

R-state hemoglobin bound to heterotropic effectors: models of the DPG, IHP and RSR13 binding sites

We performed a docking study followed by a 500-ps molecular dynamics simulation of R-state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3-diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2-[4-[(3,5-dichlorophenylcarbamoyl)-]methyl]-phenoxy]-2-methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T-state HbA, the effector binding sites are also located in the central cavity of the R-state and differ depending on effector anionic character. DPG and IHP bind between the α-subunits and the RSR13 site spans the α 1-, α 2- and β 2-subunits. The generated models provide the first report of the molecular details of R-state HbA bound to heterotropic effectors.

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.

Regulation of Inositol Phospholipid Binding and Signaling through Syndecan-4

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.

Conformational differences in CO derivatives of HbA, HbC (Eβ6K) and HbS (Eβ6V) in the presence and absence of inositol hexaphosphate detected using ultraviolet resonance Raman spectroscopy

The 229 nm excited ultraviolet resonance Raman spectra of the carbon monoxide saturated derivatives of adult human hemoglobin (HbA), HbC (Eβ6K) and HbS (Eβ6V) in the presence and absence of inositol hexaphosphate (IHP) at pH 6.35 reveal a distinct pattern of local and global conformational differences. In the absence of IHP, the protein specific spectral differences are all in the form of intensity changes. The intensity differences in the W3 band indicate that, compared with HbA, the mutant Hbs have the β-chain A helix more tightly packed against the E helix, with HbS having the largest change. Intensity differences in other Raman bands indicate that the local perturbation associated with the β6 substitution propagates to other regions of the globin. The IHP-induced changes are consistent with two types of effects, a further tightening of the packing of the A helix against the E helix and the appearance of T state features indicative of a strained or weakened R quaternary conformation. The protein specificity of these effects can be accounted for by a model in which the linkage of the A and H helices of the β-subunits through the Gluβ7-Lysβ132 (H10) salt bridge is modulated by the β6-sensitive packing of the A helix against the E helix. This mechanism also accounts for the protein specificity of the IHP effects since Lysβ132 is linked to two of the residues that form the central cavity IHP binding site. © 1998 John Wiley & Sons, Ltd.

Calorimetric studies of inositol hexaphosphate binding to aquomethemoglobin A

Inositol hexaphosphate (IHP) binding to aquomethemoglobin A (metHb) has been studied by flow microcalorimetry and compared to IHP interactions with carbonmonoxyhemoglobin A (COHb) and deoxyhemoglobin A (deoxyHb). At 25° and pH 6.8 in 0.05 M NaPhos containing 0.1 M NaCl and, after correction for heats of proton extraction from buffer, IHP binds to metHb, deoxyHb and COHb with enthalpies of −80.3, −104.2 and −105.9 kJ/mol IHP, respectively. None of the above processes exhibits large apparent heat capacity changes. The binding to metHb is strongly linked to proton uptake, 1.5 H + being absorbed per IHP bound at pH 6.8, with an enthalpic contribution of −21 kJ/mol H + absorbed. Inorganic anions, including chloride, phosphate and perchlorate, bind to metHb with dissociation constants in the 0.1 M range and are released upon IHP binding. At high salt concentrations 2–4 such ions are released with enthalpic contributions of +13 to +29 kJ/mol anion released. Thus observed heats of reaction represent a difference of large energetic terms, including not only intrinsic interaction of ligand with protein but also heats of proton extraction from buffer, proton uptake by protein and anion displacement by ligand, each of which can exceed 42 kJ/mol. These results are discussed in light of a possible allosteric transition accompanying IHP binding to metHbA.

A potential determinant of enhanced crystallization of Hbc: spectroscopic and functional evidence of an alteration in the central cavity of oxyHbC.

The structural basis of the crystallizing tendencies of oxyHbC (beta 6Glu-->Lys), that produces haemolytic anaemia in homozygotes, is unknown. Using a fluorescent organic phosphate analogue (8-hydroxy-1,3,6-pyrenetrisulphonate), and conventional oxygen equilibrium studies, data suggest that the binding of inositolhexaphosphate (IHP) to oxyHbC differs from HbA, indicating perturbations of the oxyHbC central cavity, which was predicted from our earlier spectroscopic findings. To define the relationship between this conformational change in oxyHbC and its tendency to crystallize, the effect of four central cavity ligands on the crystallization rate was studied: a peptide containing 11 residues from the N-terminal portion of band 3, the full cytoplasmic domain of band 3, 2,3-diphosphoglycerate and IHP. OxyHbC crystallization was accelerated by all these central cavity ligands and not by the appropriate controls. These central cavity changes become an excellent candidate for the dramatic increase in the crystallization rate of oxyHbC.

Quantification of glycosylated hemoglobin by binding of inositol hexaphosphate.

Journal Article Quantification of glycosylated hemoglobin by binding of inositol hexaphosphate. Get access G Phillipou, G Phillipou Search for other works by this author on: Oxford Academic Google Scholar C J Seaborn C J Seaborn Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 34, Issue 5, 1 May 1988, Pages 1002–1003, https://doi.org/10.1093/clinchem/34.5.1002a Published: 01 May 1988

Electron spin echo studies of hemoglobin cyanide and nitroxide derivatives

Electron spin echo envelope modulation studies are performed on human hemoglobin cyanide, hemoglobin nitroxide and hemoglobin nitroxide + inositol hexaphosphate at neutral pH. The modulation data are Fourier transformed and are analyzed in the frequency domain. The frequency components observed from hemoglobin cyanide indicate modulation from the coordinated nitrogen N-1 of the proximal imidazole (His-F8). In the case of hemoglobin nitroxide, the binding of inositol hexaphosphate causes the nitrogen N-3 of the proximal imidazole to be protonated in some of the subunits. From a comparison with other studies on these derivatives of hemoglobin, these subunits are identified as the α-subunits.

Quaternary equilibrium analysis of the imidazole methemoglobin bound with inositol hexaphosphate

The visible and proton NMR spectral responses of imidazole methemoglobin by the binding of inositol hexaphosphate were examined in the 2–40°C range. The magnitude of the ± (inositol hexaphosphate) visible differences spectrum increased and the intensity of the 33 ppm NMR peak decreased with lowering of the temperature. The NMR results were quantitatively analyzed with a simple two-state allosteric model. The results show that the T conformer fraction is 0.6 at 20°C and that the equilibrium shifts toward the T state at lower temperature. The large changes in δH and δS associated with the equilibrium suggest participation of numerous factors in the determination of the equilibrium position. The increase in the T conformer population of imidazole methemoglobin, which is pure low-spin, suggests that the appearance of the T state with decreasing temperature is not directly coupled to an increase in spin of the heme iron.

Demonstration of inositol hexaphosphate induced changes in structure at ligand binding sites in carp hemoglobin carbonyl

Infrared and 13C NMR spectra for carbonyl ligands bound to hemoglobin from carp, Cyprinus carpio , at pH 6.0 and 25°C undergo marked shifts when inositol hexaphosphate (IHP) is added to the solution. The relative intensities of the two C-0 stretch bands at 1951 and 1968 cm −1 due to two ligand site conformers are altered by IHP in the same manner as found upon lowering the pH in the absence of IHP. Both the lowering of pH and the binding of IHP enhance the 1968 cm −1 band conformer, the conformer normally of much lower stability. 13C NMR spectra indicate the C0 sites for only one type of subunit, probably the β-subunit, are altered by IHP. These findings demonstrate directly that the observed IHP-induced shift from high affinity (R) to a low affinity (T) form is accompanied by a significant change in ligand binding site structure at two of the four subunits.

Determination of the zero-field splitting constants of high-spin metalloproteins by a continuous-wave microwave saturation technique

A method is described for determining the zero-field splitting energy of high-spin metalloenzymes and proteins by a continuous-wave saturation technique with a conventional EPR spectrometer. The method requires analysis of graphical estimates of P 1 2 from saturation curves as a function of temperature. Under the condition that the signal amplitude depends only on the spin-lattice relaxation probability, the temperature dependence of P 1 2 provides an estimate of the zero-field splitting energy for spin-lattice relaxation via an Orbach process. The zero-field splitting energies of metmyoglobin and metmyoglobin fluoride are estimated at 22 and 12 cm −1, respectively, in excellent agreement with published results obtained by magnetic susceptibility and pulse saturation recovery methods. The zero-field splitting of methemoglobin is sensitive to the binding of inositol hexaphosphate. This change is shown to be consistent with the change in iron-porphyrin stereochemistry induced by binding of the organic phosphate molecule to the protein. The method can be applied to a wide variety of high-spin paramagnetic systems.

Altered heme environments in opossum and rabbit methemoglobins.

The effect of pH and inositol hexaphosphate (IHP) on the symmetry of the heme environments in opossum (Didelphius marsupialis) and new Zealand White rabbit hemoglobins has been studied using electron spin resonance (ESR). Each methemoglobin is found to contain two different heme environments as detected by the rhombicity observed in the ESR spectrum. In both cases the rhombic nature of the ESR spectrum is influenced by pH and IHP binding, although in the case of rabbit methemoglobin the high spin ESR signal disappears above pH 8.0. In both hemoglobins, amino acid alterations in the alpha-chains are known to affect the properties of the ferrous derivatives. It is concluded that these alterations also provide the basis for the ESR spectral properties observed with the methemoglobins.

Heterotropic interactions in monomeric βSH chains from human hemoglobin

The O 2 affinity of β SH chains is lowered by H +, inositol hexaphosphate (IHP), and CO 2. As the oxygen affinity of β SH monomers ( β 1 SH) is lower than that of β SH tetramers ( β 4 SH), it is possible that IHP and CO 2 exert their influence on the O 2 affinity of β SH chains by increasing the dissociation constant of β 4 SH rather than by a direct effect on the molecule. In order to test for this hypothesis we have measured the O 2 affinity of β SH chains as a function of protein concentration at various concentrations of IHP and inorganic phosphates in the absence and presence of CO 2. From these data association constants for the binding of IHP to β 1 SH and β 4 SH as well as for the equilibrium 4 β 1 SH ⇌ β 4 SH were calculated. We found that IHP and CO 2 influence the oxygen affinity of β 1 SH. It was furthermore established that inorganic phosphate enhances the stability of β 4 SH while IHP favors its dissociation in monomers.