Molecular basis of bird respiration: primary hemoglobin structure component from Tufted duck (Aythya fuligula, Anseriformes)--role of alpha99Arg in formation of a complex salt bridge network

The primary structure of the major hemoglobin component, HbA (alpha(A)- and beta-chain), from Tufted duck (Aythya fuligula) is presented. The separation of the globin subunits was achieved by ion exchange chromatography on CM-cellulose in 8 M urea. The amino acid sequence was determined by automatic Edman degradation of native chains as well as tryptic and hydrolytic peptides in a gas-phase sequencer. The automated homology model was generated by the protein structure modeling package WHAT IF using the crystal structure coordinates of Bar-headed goose hemoglobin. The 3D structure prediction enables alpha99Arg and beta101Glu to emerge as a new intersubunit contact site not found in the hemoglobin structure of any other species. alpha99Arg forms a complex salt bridge network involving alpha99Arg-beta101Glu-beta104Arg-beta108Asp. Also the substitution at alpha34 --> Ile, alpha38 --> Gln and beta55 --> Leu serves to stabilize the oxy-structure, leading to higher oxygen affinity.     

Carnivora: the primary structure of the alpha-chains of ferret (Mustela putorius furo, Mustelidae) hemoglobins

Ferret erythrocytes contain two hemoglobins differing only by their alpha-chains. The primary structure of the common beta-chain has been previously described; the complete sequence of the two alpha-chains are reported in this paper. The globin chains were separated by ion-exchange chromatography; the alpha-chains (42 steps), their tryptic peptides as well as the prolyl-peptides were subjected to automatic liquid- and gas-phase Edman degradation. The two alpha-chains are very similar, differing at only one position (Asp15----Gly15). Comparison with human hemoglobin alpha-chain shows 16 and 17 exchanges, for alpha 1 and alpha II chains, respectively; two substitutions involve alpha 1/beta 1 contacts and one the heme contacts. A high degree of homology was noted when the alpha-chains were compared to the corresponding chains of other representatives of the Carnivora order.     

Minimal structural requirements for root effect: crystal structure of the cathodic hemoglobin isolated from the antarctic fish Trematomus newnesi

The cathodic hemoglobin component of the Antarctic fish Trematomus newnesi (HbCTn) is a Root-effect protein. The interpretation of its functional properties in relation to its sequence is puzzling. Indeed, HbCTn sequence is characterized by an extremely low histidyl content, and in particular by the lack of His146beta and His69beta, which are believed to be important in Bohr and Root effects, respectively. Furthermore, previous analyses suggested that the local environment of Asp95alpha, Asp99beta, and Asp101beta should not be appropriate for the formation of Asp-Asp interactions, which are important for the Root effect. Here, we report the high-resolution crystal structure of the deoxy form of HbCTn. Our data provide a structural interpretation for the very low oxygen affinity of the protein and insights into the structural determinants of the Root effect protein. The structure demonstrates that the presence of Ile41alpha and Ser97alpha at the alpha1beta2 interface does not prevent the formation of the inter-Asp interactions in HbCTn, as previous studies had suggested. The present data indicate that the hydrogen bond formed between Asp95alpha and Asp101beta, which is stabilized by Asp99beta, is per se sufficient to generate the Root effect, and it is the minimal structural requirement needed for the design of Root-effect Hbs.     

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.     

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.     

Characteristic of aromatic amino acid substitution at alpha 96 of hemoglobin

Replacement of valine by tryptophan or tyrosine at position alpha96 of the alpha chain (alpha96Val), located in the alpha(1)beta(2) subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the alpha96 position. The characteristic of aromatic amino acid substitution at the alpha96 of hemoglobin has been further investigated by producing double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp). r Hb (alpha42Tyr --> Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between alpha42Tyr and beta99Asp in thealpha(1)beta(2) subunit interface of deoxy Hb A. The second mutation, alpha96Val -->Trp, may compensate the functional defects of r Hb (alpha42Tyr --> Phe), if the stability due to the introduction of trypophan at the alpha 96 position is strong enough to overcome the defect of r Hb (alpha42Tyr --> Phe). Double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb (alpha42Tyr --> Phe). (1)H NMR spectroscopic data of r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between alpha 42Tyr and beta 99Asp is essential for the novel oxygen binding properties of deoxy Hb (alpha96Val --> Trp) .     

The crystal structure of bar-headed goose hemoglobin in deoxy form: the allosteric mechanism of a hemoglobin species with high oxygen affinity

The crystal structure of a high oxygen affinity species of hemoglobin, bar-headed goose hemoglobin in deoxy form, has been determined to a resolution of 2.8 A. The R and R(free) factor of the model are 0.197 and 0.243, respectively. The structure reported here is a special deoxy state of hemoglobin and indicates the differences in allosteric mechanisms between the goose and human hemoglobins. The quaternary structure of the goose deoxy hemoglobin shows obvious differences from that of human deoxy hemoglobin. The rotation angle of one alphabeta dimer relative to its partner in a tetramer molecule from the goose oxy to deoxy hemoglobin is only 4.6 degrees, and the translation is only 0.3 A, which are much smaller than those in human hemoglobin. In the alpha(1)beta(2) switch region of the goose deoxy hemoglobin, the imidazole ring of His beta(2)97 does not span the side-chain of Thr alpha(1)41 relative to the oxy hemoglobin as in human hemoglobin. And the tertiary structure changes of heme pocket and FG corner are also smaller than that in human hemoglobin. A unique mutation among avian and mammalian Hbs of alpha119 from proline to alanine at the alpha(1)beta(1 )interface in bar-headed goose hemoglobin brings a gap between Ala alpha119 and Leu beta55, the minimum distance between the two residues is 4.66 A. At the entrance to the central cavity around the molecular dyad, some residues of two beta chains form a positively charged groove where the inositol pentaphosphate binds to the hemoglobin. The His beta146 is at the inositol pentaphosphate binding site and the salt-bridge between His beta146 and Asp beta94 does not exist in the deoxy hemoglobin, which brings the weak chloride-independent Bohr effect to bar-headed goose hemoglobin.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

Primary structure of the hemoglobins from Sphenodon (Sphenodon punctatus, Tuatara, Rynchocephalia). Evidence for the expression of alpha D-gene

Sphenodon is the sole representative of the "beakhead" reptiles which were widely distributed during the Triassic period before the spectacular rise of dinosaurs. Sphenodon punctatus is the only survivor ("living fossil") of this period. The morphological features of Sphenodon are remarkably conservative and differ little from reptiles living 200 million years ago. In the present paper the determination of the primary structure of the tetrameric hemoglobins is described: three components are identified: hemoglobin A' (alpha A2 beta II2), hemoglobin A (alpha A2 beta I2) and hemoglobin D (alpha D2 beta II2). The components were characterized electrophoretically, the four different peptide chains were characterized by Triton electrophoresis as well as by high-performance liquid chromatography. The hemoglobins and--under dissociating conditions--also the chains, were isolated on columns of cellulose ion exchangers. Sequence determination was carried out after cleavage of the individual chains with trypsin and after a specific chemical cleavage of the Asp-Pro bond. For sequence determination the film technique and gas-phase method were employed. The data are compared with the sequence of the human hemoglobin, and interpretations of the amino-acid sequences are given. Particularly notable is the evidence of hemoglobin D: this hemoglobin (alpha D2 beta II2) is found only in birds, and in two cases in turtles. However, this component is not found in other reptiles. The results make possible an interpretation of the relatively high oxygen affinity and explain the lack of cooperativity (myoglobin properties) of these tetrameric hemoglobins.     

Protein structure and cDNA nucleotide sequence of the Japanese quail alpha A globin.

The primary structure of the alpha polypeptide chain (alpha A) of the major component (QII) of Japanese quail hemoglobin was determined by protein and cDNA sequence analysis. The amino-acid sequences of all the soluble tryptic peptides were determined by the conventional protein sequencing technology. The sequence of the remaining portion, which contained an insoluble "core region", was determined through determination of the cDNA nucleotide sequence. The cDNA clones coding for the alpha A globin were isolated from the quail reticulocyte cDNA library, mapped by restriction enzyme digestion, and the nucleotide sequence was determined completely. The primary structure of quail alpha A globin shows a close similarity to that of chicken alpha A globin.     

The structural and functional analysis of the hemoglobin D component from chicken

Oxygen binding by chicken blood shows enhanced cooperativity at high levels of oxygen saturation. This implies that deoxy hemoglobin tetramers self-associate. The crystal structure of an R-state form of chicken hemoglobin D has been solved to 2.3-A resolution using molecular replacement phases derived from human oxyhemoglobin. The model consists of an alpha2 beta2 tetramer in the asymmetric unit and has been refined to a R-factor of 0.222 (R-free = 0.257) for 29,702 reflections between 10.0- and 2.3-A resolution. Chicken Hb D differs most from human oxyhemoglobin in the AB and GH corners of the alpha subunits and the EF corner of the beta subunits. Reanalysis of published oxygen binding data for chicken Hbs shows that both chicken Hb A and Hb D possess enhanced cooperativity in vitro when inositol hexaphosphate is present. The electrostatic surface potential for a calculated model of chicken deoxy-Hb D tetramers shows a pronounced hydrophobic patch that involves parts of the D and E helices of the beta subunits. This hydrophobic patch is a promising candidate for a tetramer-tetramer interface that could regulate oxygen binding via the distal histidine.     

Primary structure of alpha and beta chains from the major hemoglobin component of the magpie goose (Anseranas semipalmata, Anatidae)

The amino acid sequence of the alpha and beta chains from the major hemoglobin component (HbA) of Australian Magpie Goose (Anseranas semipalmata) is given. The minor component with the alpha D chains was detected, but only found in low concentrations. By homologous comparison, Greylag Goose hemoglobin (Anser anser) and Australian Magpie Goose alpha chains differ by 13 amino acids or 17 nucleotide (4 two point mutations) exchanges, beta chains by 6 exchanges. Seven alpha 1 beta 1 contacts are modified by substitutions in positions alpha 30-(B11)Glu leads to Gln, alpha 34(B15)Thr leads to Gln, alpha 35(B16)-Ala leads to Thr, alpha 36(B17)Tyr leads to Phe, beta 55(D6)Leu leads to Ile, beta 119(GH2)Ala leads to Ser and beta 125(H3)Glu leads to Asp. Further, one alpha 1 beta 2 contact point was changed in beta 39(C5)Gln leads to Glu. Mutation in this position, except in two abnormal human hemoglobins, was not found in any species. Amino acid exchanges between hemoglobin of Australian Magpie Goose and other birds are discussed.     

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.     

The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C

Dephosphorylation of SpoIIAA-P by SpoIIE is strictly dependent on the presence of the bivalent metal ions Mn2+ or Mg2+. Replacement by Ala of one of the four Asp residues, invariant in all representatives of protein phosphatase 2C, completely abolished the SpoIIE phosphatase activity in vitro, whilst replacement of the Asp residues by another acidic amino acid, Glu, had varying effects on the activities of the resulting mutated proteins. D610E and D795E exhibited some residual activity while D628E and D745E were without enzymatic activity. The results suggest that the functional model in which metal-associated water molecules are involved in the dephosphorylation reaction catalyzed by human protein phosphatase 2C alpha can also be applied to the bacterial protein phosphatase 2C-like protein.     

Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...     

Hemoglobins of reptiles. The primary structure of the major and minor hemoglobin component of adult Western Painted Turtle (Chrysemys picta bellii)

Red blood cells of adult Western Painted Turtles (Chrysemys picta bellii) contain two hemoglobin components: HbA (alpha A2 beta 2) and HbD (alpha D2 beta 2). We present the complete amino-acid sequences of the alpha A-chains from the major component and of the beta-chains common to both components. Structural features are discussed with respect to the animals extreme tolerance of severe hypoxic conditions during hibernation which is accompanied by a high oxygen affinity of the hemoglobin. The strong ATP dependence of Western Painted Turtle hemoglobin oxygen affinity is contrasted by the loss of one ATP-binding site, beta 143(H21)-Arg----Leu. The primary structure of the beta-chains excludes an allosteric control mechanism by hydrogencarbonate as it was found in crocodiles. Except in turtles a hemoglobin pattern with HbA and HbD sharing the same beta-subunits has been found only in birds. In comparison to other vertebrate hemoglobins there is a surprising similarity of the sequences to those of bird hemoglobins. alpha A- as well as alpha D-chains show larger homologies to chains of the same type in different species than alpha A- and alpha D-chains to each other in the same species. This indicates a duplication of the alpha-gene preceding the divergence of turtles and birds.     

An additional H-bond in the alpha 1 beta 2 interface as the structural basis for the low oxygen affinity and high cooperativity of a novel recombinant hemoglobin (beta L105W)

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

Chemical modification of a variant of human MIP-1alpha; implications for dimer structure

A sequence variant of human MIP-1alpha, in which Asp26 has been replaced by Al alpha, has been chemically modified by the addition of 13C-labeled methyl groups at each of the lysine residues and the N-terminus. The sites of methylation have been verified by a combination of MALDI-TOF mass spectrometric experiments and tryptic digestion followed by N-terminal mapping. The effect of the modification on the structure and activity of the protein have been determined by analytical ultra-centrifugation, 13C NMR spectroscopy and receptor binding studies. The results of these experiments suggest that huMIP-alpha D26A (BB10010), when present as a dimer, adopts a globular structure, like MCP-3, rather than the elongated or cylindrical structure determined for dimers of huMIP-1beta and RANTES.