Dimeric Hemoglobin Research Articles

Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

A heterodimer of α and β hemoglobin chains functions as an innate anticancer agent.

Metastatic (as well as tumor) microenvironments contain both cancer-promoting and cancer-restraining factors. The balance between these opposing forces determines the fate of cancer cells that disseminate to secondary organ sites. In search for microenvironmental drivers or inhibitors of metastasis, we identified, in a previous study, the beta subunit of hemoglobin (HBB) as a lung-derived antimetastatic factor. In the present study, exploring mechanisms regulating melanoma brain metastasis, we discovered that brain-derived factors restrain proliferation and induce apoptosis and necrosis of brain-metastasizing melanoma cells. Employing various purification procedures, we identified a heterodimer composed of hemoglobin alpha and beta chains that perform these antimetastatic functions. Neither the alpha nor the beta subunit alone was inhibitory. An alpha/beta chain dimer chemically purified from human hemoglobin inhibited the cell viability of primary melanomas, melanoma brain metastasis (MBM), and breast cancer cell lines. The dimer-induced DNA damage, cell cycle arrest at the SubG1 phase, apoptosis, and significant necrosis in four MBM cell lines. Proteomic analysis of dimer-treated MBM cells revealed that the dimer downregulates the expression of BRD4, GAB2, andIRS2 proteins, playing crucial roles in cancer cell sustainability and progression. Thus, we hypothesize that the hemoglobin dimer functions as a resistance factor against brain-metastasizing cancer cells.

Structural stability of R-state conformation of carbonmonoxyl sickle and normal hemoglobin dimer

A mutation at the sixth residue, glutamic acid to valine, in beta chain of hemoglobin distorts the entire shape of hemoglobin into a sickle shape. The investigation of the binding mechanisms of different chains of hemoglobin under the mutated condition can give an understanding of the molecular distortion. In this work, we have studied the binding mechanism between two chains in the dimer structure of the R-state conformation of carbonmonoxyl sickle hemoglobin and is compared with that of normal hemoglobin by using molecular dynamics simulations. The binding strength between α-chain (PROA) and β-chain (PROB) in hemoglobin dimer has been analyzed by estimating hydrogen bonds, salt bridges, hydrophobic interactions and non-bonded interactions (electrostatics and van der Waals). The quantitative estimation of aforementioned interactions depicts that the structural stability of normal hemoglobin dimer is found to be greater than that of sickle one. The outcomes of such interactions are also supported by the estimated free energy between the chains in R-state conformation of the dimers. The difference of binding free energy, calculated by utilizing the umbrella sampling technique, is found to be ≈ (0.67 ± 0.06) kcal/mol. Communicated by Ramaswamy H. Sarma

The dynamics of hemoglobin-haptoglobin complexes. Relevance for oxidative stress

Haptoglobin (Hp) is a protein with role in oxidative stress prevention, which it fulfills by trapping hemoglobin (Hb) dimers in a strongly-bound complex, thus reducing Hb's ability to engage in oxidative stress reactions which are greatly enhanced when the Hb tetramer falls apart into dimers. A first molecular dynamics (MD) analysis is presented here for the Hb-Hp pair. Also, for the first time the tyrosyl (Tyr) radicals in globins are examined with MD calculations directed at comparing with electron paramagnetic resonance (EPR)-detected parameters, with the aim of identifying the location of free radicals on the globin. Such assignments are needed in order to complete our understanding of the manner in which globins deal with oxidative stress, as well as for the ways the organism deals with the oxidative stress occasionally accidentally amplified by the globins via their reactive heme and Tyr moieties. The EPR-detected free radical in Hb and in Hb:Hp is characterized experimentally by a measurable increase in hydrophobicity upon Hb:Hp complexation and by a constant ∼53° θ angle. The x-ray structure and MD-derived hydrophobicity-related parameters appear to support Y145, Y35 and Y24 as likely locations of the Hb radical – arguing strongly against Y42, Y130 and Y140. The θ values appear to favor Y42, with the MD data nevertheless also arguing in favor of Y35 and, to a lesser extent, Y145 and Y140. Y130 and Y24 appear the least likely candidates according to the θ values, especially in view of the MD data. Overall, Y35 and Y145 appear to be the two candidates with least arguments against them for satisfying the two criteria (θ values and hydrophobicity) for matching the experimentally-observed EPR radical in Hb and Hb:Hp.

Electrostatic Interaction of Globins with Phospholipid Membranes

For the structures of sperm whale myoglobin (swMb), horse heart myoglobin (hhMb), hemoglobin I (HbI) from botfly Gasterophilus intestinalis (giHbI), and monomeric and dimeric hemoglobins HbI and HbII from Lucina pectinata mollusk (lpHbI and lpHbII), roles of electrostatic interactions in stabilization of native conformations of the heme cavity were analyzed, as well as their possible destabilization by interaction with the negatively charged phospholipid membranes. The native conformation of the heme cavity in these globins, both its proximal and distal parts, was shown to be sustained by a system of hydrogen bonds involving the proximal and distal protein residues, the heme propionates, and the nearby polar amino acids on the protein surface (His, Arg, Lys). The hydrogen bond network in the proximal part of the heme pocket controls the position of the Fe atom either outside or within the protoporphyrin plane, which affects the efficiency of ligand binding. In the distal part, the hydrogen bond network formed with the distal protein residue involved (HisE7 in swMb, hhMb, and giHbI and GlnE7 in lpHbII), should stabilize the conformation in which the protein can donate hydrogen to the O2 ligand. The hydrogen bond between the E7 distal residue and O2 ligand prevents rapid dissociation of the latter and plays an important role in the regulation of ligand affinity. The hydrogen bond networks found in the proximal and, especially, in the distal heme environments, must be disrupted when oxyglobin binds to the membrane surface. This disruption caused by interaction of negatively charged phospholipid heads with lysine or arginine residues exposed towards the membrane, altered local pH near the membrane, and the effect of negative electrostatic field of the membrane, can decrease the affinity of the protein for the O2 ligand, thus facilitating its dissociation at the physiological pO2 values in the cell.

Takashi Yonetani: A stellar biochemist, a man with dignity. The Roman connection

Takashi Yonetani: A stellar biochemist, a man with dignity. The Roman connection

ЭЛЕКТРОСТАТИЧЕСКОЕ ВЗАИМОДЕЙСТВИЕ ГЛОБИНОВ С ФОСФОЛИПИДНЫМИ МЕМБРАНАМИ

The present study aimed to analyze the role of electrostatic interactions contributing to the stability of the native conformations of the heme group in the structure of sperm whale myoglobin (SWMb), horse heart myoglobin (HHMb), hemoglobin I (HbI) from the botfly Gasterophilus intestinalis (giHbI) and monomeric and dimeric hemoglobins HbI and HbII from the mollusk Lucina pectinata (lpHbI and lpHbII) as well as investigate a possible reason of destabilization due to interaction with negatively charged phospholipid membranes. It was shown that the native conformation of the heme cavity in these globins, both in its proximal and distal sides, is sustained by a system of hydrogen bonds involving the proximal and distal protein residues, both heme propionic acid groups and the nearby polar amino acids on the protein surface (His, Arg, Lys). The hydrogen bond network in the proximal part of the heme pocket controls the position of the Fe atom outside or in the protoporphyrin plane, affecting the efficiency of ligand binding, while in the distal part of the heme cavity the hydrogen bond network formed with the participation of the distal protein residue (HisE7 in swMb, hhMb and giHbI, and GlnE7 in lpHbII) should, apparently, stabilize conformation where the protein is able to donate hydrogen to O2 ligand...

Thermodynamic Scaling of Interfering Hemoglobin Strain Field Waves.

Hemoglobin (Hgb) forms tetramers (dimerized α-β dimers), which enhance its globular stability and may also facilitate small gas molecule transport, as shown by recent all-atom Newtonian solvated simulations. Hydropathic bioinformatic thermodynamic scaling enables close comparisons of hemoglobin dimers with myoglobin and neuroglobin, and reveals many nonlocal wave-like features of strained Hgb structures at the coarse-grained amino acid level. The thermodynamic analysis employs two hydropathic scales, one describing abrupt first-order unfolding transitions, the other continuous second-order transitions. Small molecule exchange at hemes is a first-order process. Wave-like collective tetrameric features appropriate to ligand absorption and release, seen in optical experiments (short times), are identified thermodynamically at long times. Strain fields localized near hemes interfere with extended strain fields associated with dimer interfacial misfit, resulting in novel wavelength dependent dimer correlation function Fano antiresonances.

Hemoglobin inhibits albumin uptake by proximal tubule cells: implications for sickle cell disease.

Proximal tubule (PT) dysfunction, including tubular proteinuria, is a significant complication in young sickle cell disease (SCD) that can eventually lead to chronic kidney disease. Hemoglobin (Hb) dimers released from red blood cells upon hemolysis are filtered into the kidney and internalized by megalin/cubilin receptors into PT cells. The PT is especially sensitive to heme toxicity, and tubular dysfunction in SCD is thought to result from prolonged exposure to filtered Hb. Here we show that concentrations of Hb predicted to enter the tubule lumen during hemolytic crisis competitively inhibit the uptake of another megalin/cubilin ligand (albumin) by PT cells. These effects were independent of heme reduction state. The Glu7Val mutant of Hb that causes SCD was equally effective at inhibiting albumin uptake compared with wild-type Hb. Addition of the Hb scavenger haptoglobin (Hpt) restored albumin uptake in the presence of Hb, suggesting that Hpt binding to the Hb αβ dimer-dimer interface interferes with Hb binding to megalin/cubilin. BLAST searches and structural modeling analyses revealed regions of similarity between Hb and albumin that map to this region and may represent sites of Hb interaction with megalin/cubilin. Our studies suggest that impaired endocytosis of megalin/cubilin ligands, rather than heme toxicity, may be the cause of tubular proteinuria in SCD patients. Additionally, loss of these filtered proteins into the urine may contribute to the extra-renal pathogenesis of SCD.

Interfacial water effect on cooperativity and signal communication in Scapharca dimeric hemoglobin.

Cooperativity is important in controlling the biological functions of allosteric proteins. Understanding the detailed mechanisms of cooperativity and allosteric regulation in such proteins is essential to understanding their function; however, the mechanism by which allosteric proteins undergo conformational transitions to aid the ligand escape process and its relevance to interfacial water molecules is not well understood. Here, we perform molecular dynamics simulations to examine these issues in Scapharca dimeric hemoglobin. The effects of interfacial water on dimeric motion, ligand escape probability, gate function, and cross-correlation are considered. The results reveal that interfacial water exhibits an unbalanced stress distribution in the interface region, leading to a bias helix bundle motion that not only can expedite the escape of the first ligand but also can increase the interval between the escape of both ligands. Correspondingly, the gate function follows the same time scale as the F-helix movement, and the gate opening is non-stochastic; moreover, the inconsistent motion between the gate parts resembles cooperative behavior. An explicit analysis of the intersubunit communication map provides at least 14 signal transduction pathways. Our results significantly aid in understanding the role of interfacial water in manipulating cooperativity and will lead to further applications involving molecular machines.

Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry.

Haptoglobin (Hp) binds free hemoglobin (Hb) dimers to prevent negative consequences of Hb circulation in the extracellular environment. Although both monomeric Hb and myoglobin (Mb) species also present potential risks, their interactions with Hp have not been extensively studied. Mb is homologous to both the α- and β-chains of Hb and shares many conserved Hb/Hp interface residues, yet whether Hp binds Mb remains unclear. To address this, computational biology tools were used to predict the interactions required for Hp to bind monomeric globins, and the predicted association was tested using native electrospray ionization mass spectrometry (ESI-MS). The Hb/Hp crystal structure was used as the template to create molecular models of two Mb molecules bound to an Hp heterodimer (Mb2/Hp). Molecular modeling suggests that Mb can bind at the Hp α-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic β-chain residues involved in complementary electrostatic interactions with Hp correspond to residues with the opposite charge in Mb, suggesting unfavorable electrostatic Hp/Mb interactions at the β-chain binding site. As shown by native ESI-MS, isolated monomeric Hbα subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb β-chains. Native ESI-MS also confirmed that Mb can bind to Hp heterotetramers in solution with stoichiometries of 1:1 and 2:1 at physiological pH and ionic strength. The affinity of Hp for Mb appears to be diminished relative to that of Hb α-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.

Ligand and interfacial dynamics in a homodimeric hemoglobin.

The structural dynamics of dimeric hemoglobin (HbI) from Scapharca inaequivalvis in different ligand-binding states is studied from atomistic simulations on the μs time scale. The intermediates are between the fully ligand-bound (R) and ligand-free (T) states. Tertiary structural changes, such as rotation of the side chain of Phe97, breaking of the Lys96–heme salt bridge, and the Fe–Fe separation, are characterized and the water dynamics along the R-T transition is analyzed. All these properties for the intermediates are bracketed by those determined experimentally for the fully ligand-bound and ligand-free proteins, respectively. The dynamics of the two monomers is asymmetric on the 100 ns timescale. Several spontaneous rotations of the Phe97 side chain are observed which suggest a typical time scale of 50–100 ns for this process. Ligand migration pathways include regions between the B/G and C/G helices and, if observed, take place in the 100 ns time scale.

Comparison of ligand migration and binding in heme proteins of the globin family

The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins (via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.

Effects of mutations on the molecular dynamics of oxygen escape from the dimeric hemoglobin of Scapharca inaequivalvis

Like many hemoglobins, the structure of the dimeric hemoglobin from the clam Scapharca inaequivalvis is a “closed bottle” since there is no direct tunnel from the oxygen binding site on the heme to the solvent. The proximal histidine faces the dimer interface, which consists of the E and F helicies. This is significantly different from tetrameric vertebrate hemoglobins and brings the heme groups near the subunit interface. The subunit interface is also characterized by an immobile, hydrogen-bonded network of water molecules. Although there is data which is consistent with the histidine gate pathway for ligand escape, these aspects of the structure would seem to make that pathway less likely. Locally enhanced sampling molecular dynamics are used here to suggest alternative pathways in the wild-type and six mutant proteins. In most cases the point mutations change the selection of exit routes observed in the simulations. Exit via the histidine gate is rarely seem although oxygen molecules do occasionally cross over the interface from one subunit to the other. The results suggest that changes in flexibility and, in some cases, creation of new cavities can explain the effects of the mutations on ligand exit paths.

Effects of mutations on the molecular dynamics of oxygen escape from the dimeric hemoglobin of Scapharca inaequivalvis.

Like many hemoglobins, the structure of the dimeric hemoglobin from the clam Scapharca inaequivalvis is a "closed bottle" since there is no direct tunnel from the oxygen binding site on the heme to the solvent. The proximal histidine faces the dimer interface, which consists of the E and F helicies. This is significantly different from tetrameric vertebrate hemoglobins and brings the heme groups near the subunit interface. The subunit interface is also characterized by an immobile, hydrogen-bonded network of water molecules. Although there is data which is consistent with the histidine gate pathway for ligand escape, these aspects of the structure would seem to make that pathway less likely. Locally enhanced sampling molecular dynamics are used here to suggest alternative pathways in the wild-type and six mutant proteins. In most cases the point mutations change the selection of exit routes observed in the simulations. Exit via the histidine gate is rarely seem although oxygen molecules do occasionally cross over the interface from one subunit to the other. The results suggest that changes in flexibility and, in some cases, creation of new cavities can explain the effects of the mutations on ligand exit paths.

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin

Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9μM−1 s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.

Time-resolved crystallography for protein structure: the case of heme proteins

Time-resolved Laue diffraction is an elegant approach by which polychromatic X-ray pulses are used in combination with rapid laser triggering of light-sensitive samples so as to make a movie of protein reaction dynamics in real time. The current state-of-the-art with protein Laue diffraction has been time-resolved studies of light-induced conformational changes in myoglobin-CO with 150 ps resolution. Myoglobin has traversed the whole history of pump–probe Laue diffraction, yielding a massive amount of data that have provided considerable insight into the understanding of protein dynamics. A picture of factors governing myoglobin dynamics following ligand dissociation has been drawn from data arising from wild-type and mutant proteins. Other heme proteins have been studied using this approach: Scapharca inequivalvis dimeric hemoglobin, the FixL PAS domain and the photosynthetic reaction center complex of Blastochloris viridis.

Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: Functional and antioxidative implications

The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb–Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb–Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb–Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb–Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation.

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.