Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.     

Synthesis and characterization of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid, a new fluorescent analogue of diphosphoglyceric acid

The synthesis of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid (DIPANS) is described. It entails the synthesis of 2,4-diphosphobenzaldehyde from the action of POCl3 on 2,4-dihydroxybenzaldehyde. This is followed by coupling of the 2,4-diphosphobenzaldehyde to 1-amino-5-naphthalenesulfonic acid. Subsequent reduction with NaBH4 yields the desired product. The DIPANS exhibits an excitation maximum at 337 nm and a fluorescence emission maximum at 504 nm. This dye is quantitatively displaced by inositol hexaphosphate and is an effective analogus of diphosphoglyceric acid (DPG), possessing a KD at pH 7.0 in 0.05 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (bis-Tris) plus 0.1 M chloride of 6.88 microgram, with 1.0 molecule bound/hemoglobin tetramer. Like DPG its binding to deoxyhemoglobin decreases with increasing pH; in the presence of 0.1 M chloride it binds 0.031 times as tightly to CO hemoglobin and it yields a value for free energy coupling of 2.0 kcal/mol. The presence of 1 mM DIPANS decreases the affinity of hemoglobin for oxygen in the absence of salt from p1/2 of 0.8 mm Hg to 12.4 mm Hg. Using DPG as a competitor of DIPANS binding, a dissociation constant of 11.4 micrometer was calculated for DPG binding to deoxy-Hb at pH 7.0 in the presence of 0.05 M bis-Tris and 0.1 M chloride.     

Binding of alcohols to the peptide CO group of poly-L-proline in the I and II conformation. II. Binding constants from infrared measurements in solution

Trifluoroethanol, benzyl alcohol, and n-butanol bind to the peptide and acelyl CO groups of poly-O-acetyl-L -hydroxyproline in dichloromethane via hydrogen bonds. The binding aflinity decreases from trifhioroelhanol to n-buitanol. For the acelyl CO groups the binding does not depend on the conformation of the polymer but for the peptide CO groups the binding constants are larger by a factor of two to five time when it is in the helix II conformation (all peptide bonds trans) than when it assumes the helix I conformation (all peptide bonds cis). This preference is explained by the higher accessibility of the peptide CO groups in the II helix. The small additional energy which results from the preferential binding is sufficient, to induce a complete I → II transition because of the very high cooperativily of the system. The quantitative dependence of the equilibrium constant s for the propagation step of the transition on solvent composition (ratio of trifluoroethanol or benzyl alcohol to n-butanol) is derived from the binding data. It agrees satisfactorily with the empirical relation obtained from a best fit to transition curves of Ganseret al. The I ? II conversion of poly-L -proline is therefore an example of a conformational transition whose solvent dependence can be explained by a binding mechanism.     

Localization of binding sites for atrial natriuretic factor and angiotensin II in the central nervous system of the clawed toad Xenopus laevis

Summary The distribution of binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) was investigated in the central nervous system (CNS) of the clawed toad Xenopus laevis by means of in vitro autoradiography using [¹²⁵I]-rat ANF(99–126) or [¹²⁵I] [Val⁵] A II and [¹²⁵I]human A II as labeled ligands. The highest densities of specific ANF-binding were detected in the nucleus habenularis, thalamic regions, hypophyseal pars nervosa and nucleus interpeduncularis. Moderate ANF-binding was found in the bulbus olfactorius, pallium, septum, striatum, lateral forebrain bundle, nucleus infundibularis, hypophyseal pars distalis and tectum. The highest levels of specific A II binding sites were observed in the nucleus praeopticus, nucleus habenularis, hypophyseal pars nervosa and pars distalis, whereas the amygdala contained moderate A II binding. The existence of specific binding sites for ANF and A II in the CNS of Xenopus laevis suggests that both peptides act as neurotransmitters or neuromodulators in the amphibian CNS. The co-localization of dense binding sites for both peptides in the nucleus habenularis, hypophyseal pars nervosa and pars distalis supports the view that ANF and A II have opposite regulatory functions in these regions.     

Isolation and characterization of a transforming growth factor-beta Type II receptor cDNA from Xenopus laevis

Transforming Growth Factor-beta (TGF-beta) and their receptors have been characterized from many organisms. Two TGF-beta signaling receptors called Type I and II have been described for various ligands of the superfamily from organisms ranging from Drosophila to humans. In Xenopus laevis, TGF-beta2 and 5 have been reported and presumably, play important roles during early development. Several Type I and type II receptors for many ligands of the TGF-beta superfamily except TGF-beta type II receptor (TbetaIIR), have been characterized in Xenopus laevis. A chemical cross linking experiment using iodinated TGF-beta1 and -beta5, revealed four specific binding proteins on XTC cells. In order to understand the TGF-beta involvement during Xenopus development, a TGF-beta type II receptor (XTbetaIIR) has been isolated from a XTC cDNA library. XTbetaIIR was a partial cDNA lacking a portion of the signal peptide. The sequence analysis and homology comparison with the human TbetaIIR revealed 67% amino acid similarity in the extra cellular domain, 60% similarity in the transmembrane domain and 87% similarity in the cytoplasmic kinase domain, suggesting that XTbetaIIR is a putative TGF-beta type II receptor. In addition, the consensus amino acid motif for serine threonine receptor kinases was also present. Further, a dominant negative expression construct lacking the cytoplasmic kinase domain (engineered with the signal peptide from human TGF-beta type II receptor), was able to abolish TGF-beta mediated induction of a luciferase reporter plasmid, in a transient cell transfection assay. This substantiates the notion that XTbetaIIR cDNA can act as a receptor for TGF-beta. RT-PCR analysis using RNA isolated from various developmental stages of Xenopus laevis revealed expression of this gene in all the early stages of development and in the adult organs, except in stages 46/48.     

Comparative structural and functional studies of avian and mammalian hemoglobins

Thermal stabilities of chicken, grey lag goose (Anser anser), turkey as avian hemoglobins (Hbs); and human, bovine, sheep and horse as mammalian Hbs in hemolysate form were investigated and compared with oxygen affinities taken from literature. The thermal stability was obtained from thermal profiles using temperature scanning spectrophotometry. The buffer conditions were 50 mM Tris, pH 7.2, and 1 mM EDTA. The average of the inverse temperature transitions, average hydrophobicity, total van der Waals volume, partial molal volume and hydration potential were calculated by computational methods. The hemolysed avian Hbs have a lower oxygen affinity, higher thermal stability and higher self association than the mammalian Hbs. These observations are based on amino-acid composition, influence of ionic effectors, and the presence of Hb D in several avian Hbs. The results indicate that the avian Hbs have a more tense (T) conformation than the mammalian Hbs.     

Human Sco1 and Sco2 function as copper-binding proteins

The function of human Sco1 and Sco2 is shown to be dependent on copper ion binding. Expression of soluble domains of human Sco1 and Sco2 either in bacteria or the yeast cytoplasm resulted in the recovery of copper-containing proteins. The metallation of human Sco1, but not Sco2, when expressed in the yeast cytoplasm is dependent on the co-expression of human Cox17. Two conserved cysteines and a histidyl residue, known to be important for both copper binding and in vivo function in yeast Sco1, are also critical for in vivo function of human Sco1 and Sco2. Human and yeast Sco proteins can bind either a single Cu(I) or Cu(II) ion. The Cu(II) site yields S-Cu(II) charge transfer transitions that are not bleached by weak reductants or chelators. The Cu(I) site exhibits trigonal geometry, whereas the Cu(II) site resembles a type II Cu(II) site with a higher coordination number. To identify additional potential ligands for the Cu(II) site, a series of mutant proteins with substitutions in conserved residues in the vicinity of the Cu(I) site were examined. Mutation of several conserved carboxylates did not alter either in vivo function or the presence of the Cu(II) chromophore. In contrast, replacement of Asp238 in human or yeast Sco1 abrogated the Cu(II) visible transitions and in yeast Sco1 attenuated Cu(II), but not Cu(I), binding. Both the mutant yeast and human proteins were nonfunctional, suggesting the importance of this aspartate for normal function. Taken together, these data suggest that both Cu(I) and Cu(II) binding are critical for normal Sco function.     

A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label.

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.     

Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force

We have characterized the interaction of the Neisseria meningitidis TonB-dependent receptor HpuAB with haemoglobin (Hb). Protease accessibility assays indicated that HpuA and HpuB are surface exposed, HpuB interacts physically with HpuA, and TonB energization affects the conformation of HpuAB. Binding assays using [125I]-Hb revealed that the bipartite receptor has a single binding site for Hb (Kd 150 nM). Competitive binding assays using heterologous Hbs revealed that HpuAB Hb recognition was not species specific. The binding kinetics of Hb to HpuAB were dramatically altered in a TonB- mutant and in wild-type meningococci treated with the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that TonB and an intact proton motive force are required for normal Hb binding and release from HpuAB. Our results support a model in which both HpuA and HpuB are required to form a receptor complex in the outer membrane with a single binding site, whose structure and ligand interactions are significantly affected by the TonB-mediated energy state of the receptor.     

Crystal structure of the human TbetaR2 ectodomain--TGF-beta3 complex

Transforming growth factor-beta (TGF-beta) is the prototype of a large family of structurally related cytokines that play key roles in maintaining cellular homeostasis by signaling through two classes of functionally distinct Ser/Thr kinase receptors, designated as type I and type II. TGF-beta initiates receptor assembly by binding with high affinity to the type II receptor. Here, we present the 2.15 A crystal structure of the extracellular ligand-binding domain of the human TGF-beta type II receptor (ecTbetaR2) in complex with human TGF-beta3. ecTbetaR2 interacts with homodimeric TGF-beta3 by binding identical finger segments at opposite ends of the growth factor. Relative to the canonical 'closed' conformation previously observed in ligand structures across the superfamily, ecTbetaR2-bound TGF-beta3 shows an altered arrangement of its monomeric subunits, designated the 'open' conformation. The mode of TGF-beta3 binding shown by ecTbetaR2 is compatible with both ligand conformations. This, in addition to the predicted mode for TGF-beta binding to the type I receptor ectodomain (ecTbetaR1), suggests an assembly mechanism in which ecTbetaR1 and ecTbetaR2 bind at adjacent positions on the ligand surface and directly contact each other via protein--protein interactions.     

Interaction of copper(II) with hemoglobins in the unliganded conformation

The interaction of exogenous Cu(II) with stable T-state Ni(II)- and Cu(II)-reconstituted hemoglobins has been studied. The relative binding affinities for the two human hemoglobin Cu(II) binding sites are found to be reversed in these hemoglobins relative to native iron(II) hemoglobin A. Nickel hemoglobin, modified by N-ethylmaleimide (NEM), iodoacetamide, and carboxypeptidase A, is used to establish that the observed differences can be attributed to the protein quaternary conformation and not to the metal substitution. Magnetic interactions between the Cu(II) responsible for oxidation and the metal-heme center suggest that the Cu(II) is closer to the heme in T-state hemoglobin than R-state hemoglobin. This finding suggests a pathway for T-state heme oxidation which does not require the beta-93 sulfhydryl group, consistent with rapid Cu(II) oxidation for NEM-reacted deoxyhemoglobin.     

Control of the allosteric equilibrium of hemoglobin by cross-linking agents

The kinetics of ligand rebinding have been studied for modified or cross-linked hemoglobins (Hbs). Several compounds were tested that interact with alpha Val 1 or involve a cross-link between alpha Val 1 and alpha Lys 99 of the opposite dimer. By varying the length of certain cross-linking molecules, a wide range in the allosteric equilibrium could be obtained. Several of the mono-aldehyde modified Hbs show a shift toward the high affinity conformation of Hb. At the other extreme, for certain di-aldehyde cross-linked Hbs, the CO kinetics are typical of binding to deoxy Hb, even at low photodissociation levels, with which the dominant photoproduct is the triply liganded species; in these cases the hemoglobin does not switch from the low to high affinity state until after the fourth ligand is bound. Although each modified Hb shows only two distinct rates, the kinetic data as a function of dissociation level cannot be simulated with a simple two-state model. A critical length is observed for the maximum shift toward the low affinity T-state. Longer or shorter lengths of the cross-linker yielded more high affinity R-state. Unlike native Hb, which is in equilibrium with free dimers, the cross-linked Hbs maintain the fraction slow kinetics, which is unique to Hb tetramers, even at 0.5 microM (total heme). Addition of HbCN to unmodified HbCO solutions results in dimer exchange, which decreases the relative fraction of slow bimolecular kinetics; the cross-linked Hbs did not show such an effect, indicating that they do not participate in dimer exchange.     

Isolation and cDNA-derived amino acid sequences of hemoglobin and myoglobin from the deep-sea clam Calyptogena kaikoi

The heterodont clam Calyptogena kaikoi, living in the cold-seep area at a depth of 3761 m of the Nankai Trough, Japan, has abundant hemoglobins and myoglobins in erythrocytes and adductor muscle, respectively. Two types of hemoglobins (Hb I and Hb II) were isolated, and the complete amino acid sequences of Hb I (145 residues) and Hb II (137 residues) were obtained with combination of cDNA and protein sequencing. The amino acid sequences of C. kaikoi Hbs I and II differed from homologous chains of the congeneric clam Calyptogena soyoae in eight and five positions, respectively. The distal (E7) His, one of the functionally important residues in hemoglobin and myoglobin, was replaced by Gln in hemoglobins of C. kaikoi. A phylogenetic analysis of clam hemoglobins indicates that the evolutionary rate of Calyptogena hemoglobins is rather faster than those of other clams, suggesting that the mutation rate might be accelerated in the deep-sea animals around the areas of cold seeps or hydrothermal vents. On the other hand, it was found unexpectedly that two myoglobins Mbs I and II, isolated from the red adductor muscle, are identical in amino acid sequence Hbs I and II, respectively. Thus it was assumed that genes for Hbs I and II are also expressed in the muscle of C. kaikoi in substitution for myoglobin gene. This suggests that the major physiological role of globins in C. kaikoi is storage of oxygen under the low oxygen conditions, rather than circulating of oxygen.     

Conformational constraint of mammalian,chicken, and salmon GnRHs,but not GnRH II,enhances binding at mammalian and nonmammalian receptors: evidence for preconfiguration of GnRH II

Mammalian GnRH (mGnRH) is believed to interact with mGnRH type I receptors in a beta-II' turn conformation involving residues 5-8. This conformation can be constrained by substitution of a D-amino acid at position 6 or by a lactam ring involving residues 6 and 7, thereby increasing receptor binding affinity. It has been proposed that this is not the case for non-mGnRH receptors. However, we show that this conformational constraint increases the binding affinity of mammalian, chicken, and salmon GnRH for the chicken and catfish receptors, as well as for the mouse receptor. Therefore, we conclude that the beta-II' turn conformation enhances ligand binding for non-mGnRH as well as mGnRH type I receptors. In contrast, most substitutions of a D-amino acid in position 6 have limited effect on binding affinity for GnRH II. We suggest that this ligand is preconfigured through intramolecular interactions, which accounts for its high binding affinity and total conservation of primary structure over 500 million years of evolution.     

Rational design of inhibitors that bind to inactive kinase conformations

The majority of kinase inhibitors that have been developed so far--known as type I inhibitors--target the ATP binding site of the kinase in its active conformation, in which the activation loop is phosphorylated. Recently, crystal structures of inhibitors such as imatinib (STI571), BIRB796 and sorafenib (BAY43-9006)--known as type II inhibitors--have revealed a new binding mode that exploits an additional binding site immediately adjacent to the region occupied by ATP. This pocket is made accessible by an activation-loop rearrangement that is characteristic of kinases in an inactive conformation. Here, we present a structural analysis of binding modes of known human type II inhibitors and demonstrate that they conform to a pharmacophore model that is currently being used to design a new generation of kinase inhibitors.     

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor

The angiotensin II type I (AT(1)) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn(111) of the AT(1) receptor constitutively activates the receptor leading to Gq-coupled IP(3) production independent of Ang II binding. The Ang II-activated conformation of the AT1(N111G) receptor was proposed to be similar to that of the wild-type AT(1) receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT(1) receptors are different. Upon Ang II binding an activated conformation of the wild-type AT(1) receptor activates G protein and recruits beta-arrestin. In contrast, the agonist-bound AT1(N111G) mutant receptor preferentially couples to Gq and is inadequate in beta-arrestin recruitment.     

Hemoglobins of the Lucina pectinata/bacteria symbiosis. II. An electron paramagnetic resonance and optical spectral study of the ferric proteins

We report an optical and EPR spectral study of three hemoglobins, Hb I, II, and III, from the gill of the clam Lucina pectinata. Hemoglobin I reacts much more avidly with hydrogen sulfide than do Hbs II and III. The proximal ligand to the heme iron of each hemoglobin is histidyl imidazole. The acid/alkaline transition of ferric Hb I occurs with pK 9.6; those of ferric Hbs II and III with pK 6.6 and 5.9, respectively. At their acid limits each ferric hemoglobin exists as aquoferric hemoglobin. Broadening of the g = 6 resonance suggests that the bound water enjoys great positional freedom. Ferric Hb I, at the alkaline limit (pH 11), exists as ferric hemoglobin hydroxide. Ferric Hbs II and III, at their alkaline limit (pH 7.5), each exist as equal mixtures of two species. The low spin species with optical maxima near 541 and 576 nm and g values of 2.61, 2.20, and 1.82, are identified as ferric hemoglobin hydroxide. The high spin species, with optical maxima near 486 and 603 nm and g values of 6.71, 5.87, and 5.06, resemble Dicrocoelium hemoglobin and hemoglobin MSaskatoon. Here we show that Hbs II and III resemble hemoglobin MSaskatoon in which a distal tyrosinate oxygen ligated to the ferric heme iron at alkaline pH is displaced by water at acid pH. The H2S product of ferric Hb I is identified as ferric hemoglobin sulfide.     

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.