Reversible hexacoordination of alpha-hemoglobin-stabilizing protein (AHSP)/alpha-hemoglobin Versus pressure. Evidence for protection of the alpha-chains by their chaperone

Using high hydrostatic pressure or hydrogen peroxide as perturbing agents, we demonstrate a protective effect of the chaperone AHSP for the alpha-chains of Hb. High pressure induces an irreversible aggregation of the ferrous deoxy alpha-chains, whereas the AHSP/alpha-Hb complex shows reversible hexacoordination of the alpha-Hb without protein aggregation. Upon pressure release, the relaxation kinetics of the transition from the hexacoordinated to pentacoordinated form of alpha-Hb in the presence of AHSP exhibit a biphasic shape. High pressure did not induce dissociation of alpha-Hb from its chaperone, as evidenced by the ligand binding kinetics that show a unique rate for the AHSP/alpha-Hb complex. For both free alpha-Hb and the AHSP/alpha-Hb complex, the bimolecular rate constant of CO binding (k(CO)(on)) versus pressure exhibits a bell shape, attributed to the transition of the rate-determining step from the chemical barrier to the migration of CO within the protein matrix. These results reveal a plasticity of the alpha-Hb active site in the presence of the chaperone and indicate that the AHSP was still active at 300 MPa. The ferric state of the AHSP/alpha-Hb complex shows hexacoordination even at atmospheric pressures, indicating a His-Fe-His binding scheme as previously observed in neuroglobin and cytoglobin. The reaction with hydrogen peroxide of ferric alpha-Hb within the complex also demonstrates a protection against aggregation.     

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.     

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.     

GATA-1 and Oct-1 are required for expression of the human alpha-hemoglobin-stabilizing protein gene

Alpha-hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds and stabilizes alpha-hemoglobin during normal erythropoiesis and in pathological states of alpha-hemoglobin excess. AHSP has been proposed as a candidate gene in some Heinz body hemolytic anemias and as a modifier gene in the beta-thalassemia syndromes. To gain additional insight into the molecular mechanisms controlling the erythroid-specific expression of the AHSP gene and provide the necessary tools for further genetic studies of these disorders, we have initiated identification and characterization of the regulatory elements controlling the human AHSP gene. We mapped the 5'-end of the AHSP erythroid cDNA and cloned the 5'-flanking genomic DNA containing the putative AHSP gene promoter. In vitro studies using transfection of promoter/reporter plasmids in human tissue culture cell lines, DNase I footprinting analyses and gel mobility shift assays, identified an AHSP gene erythroid promoter with functionally important binding sites for GATA-1- and Oct-1-related proteins. In transgenic mice, a reporter gene directed by a minimal human AHSP promoter was expressed in bone marrow, spleen, and reticulocytes, but not in nonerythroid tissues. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of the promoter region and occupancy by GATA-1. The AHSP promoter is an excellent candidate region for mutations associated with decreased AHSP gene expression.     

Molecular mechanism of AHSP-mediated stabilization of alpha-hemoglobin

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.     

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

Background

AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).

Methods

Recombinant AHSP, WT α-Hb:AHSP and α-Hb^HE7Q:AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.

Results

AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.

Conclusion

α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.

General significance

The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia.     

Biophysical characterization of the alpha-globin binding protein alpha-hemoglobin stabilizing protein

Alpha-hemoglobin stabilizing protein (AHSP) is a small (12 kDa) and abundant erythroid-specific protein that binds specifically to free alpha-(hemo)globin and prevents its precipitation. When present in excess over beta-globin, its normal binding partner, alpha-globin can have severe cytotoxic effects that contribute to important human diseases such as beta-thalassemia. Because AHSP might act as a chaperone to prevent the harmful aggregation of alpha-globin during normal erythroid cell development and in diseases of globin chain imbalance, it is important to characterize the biochemical properties of the AHSP.alpha-globin complex. Here we provide the first structural information about AHSP and its interaction with alpha-globin. We find that AHSP is a predominantly alpha-helical globular protein with a somewhat asymmetric shape. AHSP and alpha-globin are both monomeric in solution as determined by analytical ultracentrifugation and bind each other to form a complex with 1:1 subunit stoichiometry, as judged by gel filtration and amino acid analysis. We have used isothermal titration calorimetry to show that the interaction is of moderate affinity with an association constant of 1 x 10(7) m(-1) and is thus likely to be biologically significant given the concentration of AHSP (approximately 0.1 mm) and hemoglobin (approximately 4 mm) in the late pro-erythroblast.     

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O₂ bond, explaining previous reports that AHSP decreases αHb O₂ affinity roughly 4-fold and promotes autooxidation due primarily to a 3–4-fold increase in the rate of O₂ dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O₂ dissociation rate and equilibrium constants, and reduced O₂-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.     

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant,AHSPV56G

A kinetic analysis has been made of the interaction of α-Hb chains with a mutant α-hemoglobin stabilizing protein, AHSP^V56G, which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent α chains until final binding to the partner β-Hb. Rather than protecting α chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with α-Hb. For both AHSP^WT and AHSP^V56G, the binding to α-Hb is quite rapid relative to the α-β reaction, as expected because the chaperone binding must be quite competitive to complete the α chain folding process before α-Hb binds irreversibly to β-Hb. The main kinetic difference is a dissociation rate of AHSP^V56G·α-Hb some four times faster relative to AHSP·α-Hb. Considering a role of protein folding, the AHSP^V56G apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP·α-Hb + β-Hb → AHSP + αβ) can be quite long, especially if there is an excess of AHSP relative to β-Hb monomers.     

Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

Biochemical fates of alpha hemoglobin bound to alpha hemoglobin-stabilizing protein AHSP

Alpha hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds free alpha hemoglobin (alphaHb) to maintain its structure and limit its pro-oxidant activity. Prior studies have defined two different alphaHb.AHSP complexes. Binding of AHSP to Fe(II) alphaHb induces an unusual configuration in which the F helix of the globin becomes disordered and the heme ring becomes solvent-exposed. Over time, this intermediate oxidizes to form a stable hemichrome in which the proximal (F8) and distal (E7) histidines are coordinated to the heme iron atom. The addition of betaHb to either Fe(II) or Fe(III) alphaHb.AHSP displaces AHSP to generate tetrameric (alpha(2)beta(2)) HbA species. The biochemical properties and in vivo significance of the two alphaHb.AHSP complexes are poorly understood. Here we show that Fe(III) alphaHb.AHSP forms from auto-oxidation of oxygenated alphaHb bound to AHSP and that this process is greatly accelerated at physiologic temperature and oxygen pressures. In contrast to free Fe(III) alphaHb hemichromes, AHSP-bound Fe(III) alphaHb does not precipitate and can be recycled into functional HbA. This requires enzymatic reduction of AHSP-bound alphaHb, either prior to or after extraction by beta subunits. In contrast, reaction of Fe(II) alphaHb-AHSP with betaHb generates functional HbA directly. Our findings support a model in which AHSP can either stabilize alphaHb transiently en route to HbA formation during normal erythropoiesis or convert excessive free alphaHb into a more chemically inert state from which recovery of alphaHb is possible by redox cycling.     

Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSP^WT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain.     

DNA-binding properties of gene-5 protein encoded by bacteriophage M 13. 1. The kinetics of the dissociation of gene-5-protein.polynucleotide complexes upon addition of salt

The irreversible dissociation kinetics of complexes of M13-encoded gene-5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped-flow experiments. A linear decay was found for all gene-5-protein.poly(dA) complexes and for the gene-5-protein.M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single-exponential decay was found for the dissociation of gene-5-protein.M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel is not maintained. The binding of gene-5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA-binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide. The absence of line broadening for gene-5-protein.polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.     

Ligand-induced monomerization of <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>′ studied using native mass spectrometry and fluorescence resonance energy transfer

Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.     

Dynamics of trigger factor interaction with translating ribosomes

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.     

Secondary structure mapping of DnaK-bound protein fragments: chain helicity and local helix unwinding at the binding site

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.     

Substrates modulate the rate-determining step for CO binding in cytochrome P450cam (CYP101). A high-pressure stopped-flow study.

The high-pressure stopped-flow technique is applied to study the CO binding in cytochrome P450cam (P450cam) bound with homologous substrates (1R-camphor, camphane, norcamphor and norbornane) and in the substrate-free protein. The activation volume DeltaV # of the CO on-rate is positive for P450cam bound with substrates that do not contain methyl groups. The kon rate constant for these substrate complexes is in the order of 3 x 10(6) M(-1) x s(-1). In contrast, P450cam complexed with substrates carrying methyl groups show a negative activation volume and a low kon rate constant of approximately 3 x 10(4) M(-1) x s(-1). By relating kon and DeltaV # with values for the compressibility and the influx rate of water for the heme pocket of the substrate complexes it is concluded that the positive activation volume is indicative for a loosely bound substrate that guarantees a high solvent accessibility for the heme pocket and a very compressible active site. In addition, subconformers have been found for the substrate-free and camphane-bound protein which show different CO binding kinetics.