Α2β2 Tetramer Research Articles

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

Tissue-specific regulation of the Na, K-ATPase by the cytosolic NaAF: some thoughts on brain function

The dual topology P-2 ATPase, which consists of a α²β² tetramer, explains numerous functions of the cation transporting ATPase system. The ubiquitous cytosolic protein regulator (NaAF) of 170 k Da mass regulates P-2 ATPase function in a low Ca (µM) neighborhood where Ca acts as the terminal regulator in the intracellular signaling cascade. The Na, K- ATPase also seems to function as an H, K-ATPase or a Ca-ATPase in altered states based on the local environment (low pH or high Ca) in a tissue specific manner. These altered effects are analogous to that of the 80 k Da cytosolic HAF in regulating the gastric H, K-ATPase system of the parietal cells. However there are some important differences. The HAF stimulates the Na, K-ATPase but the NaAF cannot stimulate H, K-ATPase. Also, HAF is as effective as NaAF in stimulating the kidney Na, K-ATPase but about 60% as effective in stimulating brain Na, K-ATPase. These observations reveal that the Na, K- ATPase systems from kidney and brain, consisting of different kinds of αβ–isoforms, interact differently with the HAF molecule; thus substantiating that P-2 ATPase system plays different roles in different tissues in response to an universal NaAF. Another rare feature of the HAF is that it has histone kinase activity, suggesting that the HAF and NaAF may be capable of sending a direct signal to the nucleus for gene expression.In this paper, the central role of the NaAF-regulated Na, K-ATPase system in the activity and function of brain tissue is discussed. It is noted that the altered function of the nerve terminus located Na, K-ATPase system works as a Ca-pump (after depolarization) and as a Na-pump (in repolarization) in alternate sequence. The possible role of Ca-sensing receptor (CaR) in the voltage gated channeling of Ca has been raised and the possibility of a dual channel Na/H antiporter (NhaA) in pH homeostasis is discussed.

Active Form of the Protein Kinase CK2 α2β2 Holoenzyme Is a Strong Complex with Symmetric Architecture

CK2 is a protein kinase essential for cell viability whose activity is altered in several cancers. Its mechanisms of regulation differ from those common to other eukaryotic protein kinases and are not entirely established yet. Here we present crystal structures of the monomeric form of the α2β2 holoenzyme that allow refining a formerly proposed structural model for activity regulation by oligomerization. Previous crystal structures of the CK2 holoenzyme show an asymmetric arrangement of the two α catalytic subunits around the obligate β2 regulatory subunits. Asymmetric α2β2 tetramers are organized in trimeric rings that correspond to inactive forms of the enzyme. The new crystal structures presented here reveal the symmetric architecture of the isolated active tetramers. The dimension and the nature of the α/β interfaces configure the holoenzyme as a strong complex that does not spontaneously dissociate in solution, in accordance with the low dissociation constant (∼4 nM).

Quantitative characterization of the compensating effects of trimethylamine-N-oxide and guanidine hydrochloride on the dissociation of human cyanmethmoglobin.

Dynamic light scattering was used to measure the extent of dissociation of human cyanmethemoglobin (HbCN) α2β2 tetramers into αβ dimers as a function of HbCN concentration in the presence of varying concentrations of guanidine hydrochloride (GuHCl) and trimethylamine-N-oxide (TMAO). It was found that increasing concentrations of GuHCl enhance the dissociation of HbCN, and that GuHCl-induced dissociation is progressively inhibited with increasing concentrations of TMAO. The effects of both cosolutes upon the free energy of HbCN dissociation are shown to be additive. The effect of TMAO on Hb dissociation is largely attributed to steric volume exclusion but is partially compensated by a small attractive interaction between TMAO and the protein.

Hemoglobin is present as a canonical α2β2 tetramer in dopaminergic neurons

Hemoglobin is the oxygen carrier in blood erythrocytes. Oxygen coordination is mediated by α2β2 tetrameric structure via binding of the ligand to the heme iron atom. This structure is essential for hemoglobin function in the blood. In the last few years, expression of hemoglobin has been found in atypical sites, including the brain. Transcripts for α and β chains of hemoglobin as well as hemoglobin immunoreactivity have been shown in mesencephalic A9 dopaminergic neurons, whose selective degeneration leads to Parkinson's disease. To gain further insights into the roles of hemoglobin in the brain, we examined its quaternary structure in dopaminergic neurons in vitro and in vivo. Our results indicate that (i) in mouse dopaminergic cell line stably over-expressing α and β chains, hemoglobin exists as an α2β2 tetramer; (ii) similarly to the over-expressed protein, endogenous hemoglobin forms a tetramer of 64kDa; (iii) hemoglobin also forms high molecular weight insoluble aggregates; and (iv) endogenous hemoglobin retains its tetrameric structure in mouse mesencephalon in vivo. In conclusion, these results suggest that neuronal hemoglobin may be endowed with some of the biochemical activities and biological function associated to its role in erythroid cells. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

THE MOLECULAR ENZYMOLOGY OF NITROGEN FIXATION

ABSTRACT The requirements for ATP and reductant during diazotrophic growth, the physicochemical properties of the nitrogenase proteins, and the involvement of Mo in different aspects of N2 fixation are reviewed. Irrespective of the source from which it is isolated, nitrogenase has very similar properties and is comprised of two O2-sensitive redox proteins. The Mo—Fe protein is an α2β2 tetramer of MW 220000, containing 2 Mo and approx. 33 ± 3 Fe and acid-labile S atoms per tetramer. The Mo atoms and approx. 40% of the Fe atoms can be extracted with N-methylformamide as a unique cofactor (FeMoco) which contains Mo, Fe and S2- in the ratios 1:8:4. Cluster extrusion and Mossbauer data are consistent with the presence of four 4Fe4S clusters in addition to two FeMoco clusters. The binding of substrate (C2H2), its reduction product C2H4, and the inhibitor CO to the Mo-Fe protein have been investigated by EPR spectroscopy. The Fe protein is γ2 dimer of MW 67000; spectroscopic and extrusion data indicate the prese...

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Elongation of the Fe–His Bond in the α Subunit Induced by Binding of the Allosteric Effector Bezafibrate to Hemoglobins

Human adult hemoglobin (HbA), possessing an α(2)β(2) tetramer structure, efficiently transports oxygen from the lungs to the tissues. The oxygen affinity of HbA has been shown to be regulated by organic phosphates such as 2,3-bisphosphoglycerate (BPG). Bezafibrate (BZF) is also known to alter the oxygen affinity of HbA through a mechanism largely different from that of BPG. The interaction of HbA with BZF has been characterized by (1)H NMR to elucidate the molecular mechanism responsible for the functional alteration of HbA. Paramagnetically shifted heme methyl proton signals of only the α subunit of met-azido HbA exhibited sizable downfield shifts in the presence of BZF. Since met-azido HbA exhibits the so-called thermal spin equilibrium between high and low spin states, the BZF-induced shift changes observed for the α signals can be attributed to an increase in the high spin contents in the subunit, possibly due to the elongation of the Fe-His bond.

Covalent Modifications of Hemoglobin by Nitrite Anion: Formation Kinetics and Properties of Nitrihemoglobin

The green nitrihemoglobin (α(2)β(2) tetramer, NHb) was prepared by the aerobic reaction of excess nitrite with human hemoglobin A(0) under mildly acidic conditions. A rate equation was determined and found to depend on nitrite, hydrogen ion, and oxygen concentrations: -d[HbNO(2)]/dt = [k(1) + k(2)(K(a)[HNO(2)])[O(2)](1/2)][HbNO(2)], where k(1) = (2.4 ± 0.9) × 10(-4) s(-1), k(2) = (1 ± 0.2) × 10(5) M(-5/2) s(-1), and K(a) is the acid dissociation constant for nitrous acid (4.5 × 10(-4) M). Also, the chemical properties of NHb are compared to those of the normal hemoglobin (including the addition products of common oxidation states with exogenous ligands, the alkaline transitions of the ferric forms, and the oxygen binding characteristics of the ferrous forms) and were found to be nearly indistinguishable. Therefore, the replacement of a single vinyl hydrogen with a nitro group on the periphery of each macrocycle in hemoglobin does not significantly perturb the interaction between the hemes and the heme pockets. Because nonphotochemical reaction chemistry must necessarily be most dependent on electronic ground states, it follows that the clearly visible difference in color between hemoglobin A(0) and NHb must be associated primarily with the respective electronic excited states. The possibility of NHb formation in vivo and its likely consequences are considered.

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 alpha-Hb chains with a mutant alpha-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 alpha chains until final binding to the partner beta-Hb. Rather than protecting alpha chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with alpha-Hb. For both AHSP(WT) and AHSP(V56G), the binding to alpha-Hb is quite rapid relative to the alpha-beta reaction, as expected because the chaperone binding must be quite competitive to complete the alpha chain folding process before alpha-Hb binds irreversibly to beta-Hb. The main kinetic difference is a dissociation rate of AHSP(V56G).alpha-Hb some four times faster relative to AHSP.alpha-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.alpha-Hb + beta-Hb --> AHSP + alphabeta) can be quite long, especially if there is an excess of AHSP relative to beta-Hb monomers.

Insights Into The Mechanism Of The Cobalt Containing Nitrile Hydratase From Geobacillus Pallidus

The crystal structures of the wild-type, Co(III) containing, nitrile hydratase and the mutant (βF36L / βL103S / βY127N / αD4G) enzyme from have been solved at resolutions of 1.4A and 1.15A respectively. Nitrile hydratases are noted for having an unusual cysteine claw structure at the active site. An important observation made in this paper is that cysteines α119 and α121 were both modified to cysteine sulfinic acid instead of the α121 cysteine sulfenic and α119 cysteine sulfinic acid reported previously. This was confirmed by MALDI-TOF mass spectroscopy.The nitrile hydratases catalyse the conversion of nitriles to the corresponding amides. These enzymes underlie the industrial production ofacrylamide. In the cysteine claw structure an Fe(III) or Co(III) ion is octahedrally co-ordinated to three cysteines, two of which are oxidized, and two peptide backbone amide groups. The sixth ligand, trans to the unmodified cysteine is a water molecule or a hydroxide ion. The cysteine claw has the sequence CTLCSC in the iron case and CSLCSC in the cobalt case. The enzyme itself is a α2β2 tetramer with the cysteine claw located in the α subunit and much of the active site contributed by the β subunit.Our crystal structures are almost certainly an inactive form of the enzyme, being crystallized in 100 mM MES (2[N-Morpholino]ethanesulfonic acid) which was found to be an inhibitor of the enzyme subsequent to the crystallization. However at present no evidence suggests that the inactivation is due to the oxidation of the active site cysteine sulfenic acid.The structures give insight into the determinants of the specificity of the enzyme, suggesting that βphe55 and βphe55 obstruct access to the cysteine claw complex for larger substrates allowing the hydration of only smaller aliphatic nitriles.

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.

Characterization of a Novel Caenorhabditis elegans Prolyl 4-Hydroxylase with a Unique Substrate Specificity and Restricted Expression in the Pharynx and Excretory Duct

Collagen prolyl 4-hydroxylases (C-P4Hs) have a critical role in collagen synthesis, since 4-hydroxyproline residues are necessary for folding of the triple-helical molecules. Vertebrate C-P4Hs are alpha(2)beta(2) tetramers in which the beta subunit is identical to protein-disulfide isomerase (PDI). Three isoforms of the catalytic alpha subunit, PHY-1, PHY-2, and PHY-3, have been characterized from Caenorhabditis elegans, PHY-1 and PHY-2 being responsible for the hydroxylation of cuticle collagens, whereas PHY-3 is predicted to be involved in collagen synthesis in early embryos. We have characterized transcripts of two additional C. elegans alpha subunit-like genes, Y43F8B.4 and C14E2.4. Three transcripts were generated from Y43F8B.4, and a polypeptide encoded by one of them, named PHY-4.1, assembled into active (PHY-4.1)(2)/(PDI-2)(2) tetramers and PHY-4.1/PDI-2 dimers when coexpressed with C. elegans PDI-2 in insect cells. The C14E2.4 transcript was found to have a frameshift leading to the absence of codons for two residues critical for P4H catalytic activity. Thus, C. elegans has altogether four functional C-P4H alpha subunits, PHY-1, PHY-2, PHY-3, and PHY-4.1. The tetramers and dimers containing recombinant PHY-4.1 had a distinct substrate specificity from the other C-P4Hs in that they hydroxylated poly(l-proline) and certain other proline-rich peptides, including ones that are expressed in the pharynx, in addition to collagen-like peptides. These data and the observed restricted expression of the phy-4.1 transcript and PHY-4.1 polypeptide in the pharyngeal gland cells and the excretory duct suggest that in addition to collagens, PHY-4.1 may hydroxylate additional proline-rich proteins in vivo.

Purification of a NifEN Protein Complex That Contains Bound Molybdenum and a FeMo-Co Precursor from an Azotobacter vinelandii ΔnifHDK Strain

The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.

Heterogeneous Nuclear Ribonucleoprotein-A2/B1 Modulate Collagen Prolyl 4-Hydroxylase, α (I) mRNA Stability

Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

高グリシン血症の原因となるＰタンパク質のＸ線結晶構造解析

The crystal structure of the P-protein of the glycine cleavage system from Thermus thermophilus HB8 has been determined, which reveals that P-proteins do not involve the α2 dimer universally observed in the evolutionarily related pyridoxal 5'-phosphate (PLP) -dependent enzymes. Instead, novel αβ dimers associate to form an α2β2 tetramer. The crystal structure of P-protein without PLP has also been analyzed. These structures allow the identification of functionally important residues involved in recognition of the cofactor and substrates, and show that most of these residues are well conserved among all living organisms. These results provide insights into the molecular basis of hyperglycinemia.

The Role of Alpha Hemoglobin Stabilizing Protein (AHSP) in the Formation of Hemoglobin A.

Alpha hemoglobin stabilizing protein (AHSP) binds α hemoglobin (Hb) to stabilize its structure and limit its chemical reactivity in vitro and in vivo. Addition of β Hb to α Hb-AHSP displaces AHSP to form HbA (α2β2) tetramer. Recently, we determined that binding of AHSP to free α Hb generates a novel structure in which the helix F of α Hb is disordered and the heme iron is reduced to the ferrous form and coordinated by the distal histidine (ferrous α Hb-AHSP). Over time, the heme iron within this complex oxidizes to the ferric form (ferric α Hb-AHSP) and the F helix reorders, generating a bis-histidyl heme iron that exhibits decreased capacity to catalyze the formation of reactive oxygen species. At room temperature in ambient oxygen, the conversion of ferrous to ferric α Hb-AHSP occurs over several hours. We proposed that in vivo, these complexes could serve as transient intermediates to maintain α Hb in a stable state until β Hb is available to form tetrameric HbA. The current study addresses two questions related to this hypothesis:Does bis-histidyl ferric α Hb-AHSP form more readily under physiologic conditions?Can ferrous and ferric α Hb-AHSP complexes form functional tetrameric HbA upon reaction with β Hb?The conversion of ferrous to ferric α Hb-AHSP is temperature-dependent with complete conversion occurring in 40 minutes at 37°C, as opposed to 5 hours at 22°C. Furthermore, the conversion is accelerated by partial oxygen saturation, increasing the likelihood that ferric-AHSP Hb will form in vivo. Using cellulose acetate gel electrophoresis, we demonstrated that β Hb displaces AHSP to form tetrameric HbA from both α Hb-AHSP complexes. The resulting tetramer from ferrous α Hb-AHSP is similar to HbA, both spectroscopically and in terms of oxygen binding. In contrast, the Hb tetramer formed from ferric α Hb-AHSP and β Hb is similar to HbY (αFeIII2βFeII2) spectroscopically. However, the α globin heme iron within this tetrameric complex appears to be occluded, as the rate of cyanide binding is reduced. Furthermore, it is resistant to reduction by sodium dithionite or methemoglobin reductase, suggesting that the α Hb retains its bis-histidyl nature. Together, these studies show that both α Hb-AHSP complexes are likely to form under physiological conditions. Upon interaction with β Hb, ferrous α Hb-AHSP forms functional Hb A tetramer. In contrast, ferric α Hb-AHSP is more chemically inert, but may not be able to form viable HbA.

Nucleotide-dependent Formation of Catalytically Competent Dimers from Engineered Monomeric Ribonucleotide Reductase Protein R1

Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (alpha(2)) and R2 (beta(2)), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.

Structure of P-protein of the glycine cleavage system: implications for nonketotic hyperglycinemia

The crystal structure of the P-protein of the glycine cleavage system from Thermus thermophilus HB8 has been determined. This is the first reported crystal structure of a P-protein, and it reveals that P-proteins do not involve the alpha(2)-type active dimer universally observed in the evolutionarily related pyridoxal 5'-phosphate (PLP)-dependent enzymes. Instead, novel alphabeta-type dimers associate to form an alpha(2)beta(2) tetramer, where the alpha- and beta-subunits are structurally similar and appear to have arisen by gene duplication and subsequent divergence with a loss of one active site. The binding of PLP to the apoenzyme induces large open-closed conformational changes, with residues moving up to 13.5 A. The structure of the complex formed by the holoenzyme bound to an inhibitor, (aminooxy)acetate, suggests residues that may be responsible for substrate recognition. The molecular surface around the lipoamide-binding channel shows conservation of positively charged residues, which are possibly involved in complex formation with the H-protein. These results provide insights into the molecular basis of nonketotic hyperglycinemia.