Arthur Arnone

Development of antisickling compounds that chemically modify hemoglobin S specifically within the 2,3-diphosphoglycerate binding site

Abstract In this paper we describe a class of affinity reagents that react specifically with hemoglobin S at the 2,3-diphosphoglycerate binding site and effectively disrupt the sickling process. The prototype of these compounds is the bifunctional acylating agent bis(3,5-dibromosalicyl) fumarate. Previously it was shown that this compound reacts selectively with oxyhemoglobin to cross-link the β chains of the tetramer ( Walder et al. , 1979 a ). The site of cross-linking has now been established by X-ray crystallographic studies to be from Lys82 β 1 to Lys82 β 2 , spanning the DPG ‡ binding site. The oxygen binding properties of the cross-linked hemoglobin are very similar to those of native hemoglobin A in the absence of DPG. However, in the presence of DPG, the oxygen affinity of the cross-linked hemoglobin is increased due to the blockade of the DPG site by the interchain bridge. This effect may indirectly interfere with the sickling process in vivo . More importantly the cross-link modification directly inhibits the polymerization of deoxyhemoglobin S. The solubility of the cross-linked derivative is increased by approximately 35% relative to that of native deoxyhemoglobin S. The X-ray crystallographic studies reveal that the cross-link strongly perturbs the acceptor site for Val6β in the lateral contact between molecules of deoxyhemoglobin S within the double strand of the sickle cell fiber. Our current hypothesis is that this structural perturbation weakens the lateral contact and is responsible for the observed increase in solubility. Due to the hydrophobic properties of the halogen substituents bis(3,5-dibromosalicyl) fumarate is able to traverse the red-cell membrane and, therefore, may be active in vivo . These studies provide the basis for further design and development of affinity reagents directed to the DPG binding site that may prove useful in the clinical management of sickle cell disease.

Structure of human deoxyhemoglobin specifically modified with pyridoxal compounds

Abstract Previous studies (Benesch et al., 1972, 1973, 1975) have shown that a variety of specifically modified hemoglobins having a wide range of functional properties can be prepared by reacting human hemoglobin with different pyridoxal compounds and then reducing the products with sodium borohydride. In particular, the modified hemoglobins (α PLS ‡ ‡ Abbreviations used: PLP, pyridoxal 5′-phosphate; PLS, pyridoxal 5′-sulfate; NFPLP, 2-nor-2-formylpyridoxal 5′-phosphate; DPG, 2,3-diphosphoglycerate; HbXL, αβ-NFPLP-βα hemoglobin. ) 2 β 2 , α 2 (β PLP ) 2 and αβ-NFPLP-βα have been characterized, respectively, as the reduced products of the reactions of pyridoxal 5′-sulfate with the NH2-termini of the α chains, pyridoxal 5′-phosphate with the NH2-termini of the β chains, and 2-nor-2-formylpyridoxal 5′-phosphate (which contains two reactive aldehyde groups) with one amino group from each β chain to form a stable cross-link between the β chains. In this paper, we report the three-dimensional structures of the deoxy forms of these hemoglobins as determined by X-ray diffraction methods. We find the pyridoxal side chains of these hemoglobins to be oriented as follows. 1. (1) The PLS side chains of (αPLS)2β2 are positioned so that each sulfate group replaces a weakly bound inorganic anion to form salt bridges between the newly formed secondary amine of valine 1α and the guanidinium ion of arginine 141 on the opposite α chain. The pyridoxal ring interacts with residues of the H-helix on the same α chain to which it is attached. 2. (2) In α2(βPLP)2, the PLP phosphate groups are located very near the positions occupied by the phosphate groups of 2,3-diphosphoglycerate in the deoxyhemoglobin-DPG complex, and serve as permanently bound counterions for the basic side chains of histidine 143, histidine 2, and lysine 82 of both β subunits. Each pyridoxal ring displaces a firmly bound inorganic anion and interacts with residues of the EF corner on the same β chain to which it is attached. 3. (3) In the case of αβ-NFPLP-βα, we find that the cross-link is asymmetric with the 2′ and 4′ carbons of NFPLP joined to the amino nitrogens of lysine 82β1 and valine 1β2, respectively. The phosphate group replaces the firmly bound inorganic anion mentioned above, and the 3-oxygen of the pyridoxal ring interacts with the imidazole group of histidine 143β1. Relative to the structure of deoxyhemoglobin A, no significant changes in α chain tertiary structure are observed for (αPLS)2β2, only small changes in β chain tertiary structure occur in α2(βPLP)2, but exceptionally large perturbations to the structure of one β chain result from the cross-link in αβ-NFPLP-βα. The relationships between the structures of these hemoglobin derivatives and their functional properties are discussed.

Preliminary crystallographic study of aspartate: 2-oxoglutarate aminotransferase from pig heart*

Well-ordered crystals of aspartate: 2-oxoglutarate aminotransferase have been grown by vapor diffusion from solutions of polyethylene glycol. X-ray diffraction patterns show that they belong to the orthorhombic space group P212121 with unit cell dimensions a = 124·7 , b = 130·9 , and c = 55·7 . The asymmetric unit consists of one dimer of molecular weight 92,688. The diffraction pattern extends beyond 2·8 , indicating that this crystal form is suitable for high resolution X-ray analysis.

X-ray diffraction studies of a partially liganded hemoglobin, [α(FeII-CO)β(MnII)]2

We have applied single-crystal X-ray diffraction methods to analyze the structure of [α(FeII-CO)β(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3·0 A, shows that (1) the Mn(II)-substituted β subunits are structurally isomorphous with normal deoxy β subunits, and (2) CO binding to the α subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the α heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the α1β2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.

High-resolution X-ray study of deoxy recombinant human hemoglobins synthesized from beta-globins having mutated amino termini.

The crystal structures of three mutant hemoglobins reconstituted from recombinant beta chains and authentic human alpha chains have been determined in the deoxy state at 1.8-A resolution. The primary structures of the mutant hemoglobins differ at the beta-chain amino terminus. One mutant, beta Met, is characterized by the addition of a methionine at the amino terminus. The other two hemoglobins are characterized by substitution of Val 1 beta with either a methionine, beta V1M, or an alanine, beta V1A. All the mutation-induced structural perturbations are small intrasubunit changes that are localized to the immediate vicinity of the beta-chain amino terminus. In the beta Met and beta V1A mutants, the mobility of the beta-chain amino terminus increases and the electron density of an associated inorganic anion is decreased. In contrast, the beta-chain amino terminus of the beta V1M mutant becomes less mobile, and the inorganic anion binds with increased affinity. These structural differences can be correlated with functional data for the mutant hemoglobins [Doyle, M. L., Lew, G., DeYoung, A., Kwiatkowski, L., Noble, R. W., & Ackers, G. K. (1992) Biochemistry preceding paper is this issue] as well as with the properties of ruminant hemoglobins and a mechanism [Perutz, M., & Imai, K. (1980) J. Mol. Biol. 136, 183-191] that relates the intrasubunit interactions of the beta-chain amino terminus to changes in oxygen affinity. Since the structures of the mutant deoxyhemoglobins show only subtle differences from the structure of deoxyhemoglobin A, it is concluded that any of the three hemoglobins could probably function as a surrogate for hemoglobin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Equilibrium, kinetic and structural properties of hemoglobin Cranston, an elongated β chain variant☆

Abstract Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston ‡ at 20 °C in the absence of phosphate revealed a high affinity (P50 = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Bohr effect ( −Δ log P 50 Δ pH 7–8 = 0·2 ). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P50 = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Bohr effect ( −Δ log P 50 Δ pH 7–8 = 0·2 ). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′4, of ∼ 6 × 106m−1s−1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ∼ 0·5 × 106m−1s−1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k4∼ 11 s−1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α2βAβCranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.

Site‐directed mutations of human hemoglobin at residue 35β: A residue at the intersection of the α1β1, α1β2, and α1α2 interfaces

Because Tyr35β is located at the convergence of the α1β1, α1β2, and α1α2 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 βY35F and βY35A. 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 Tyr35β 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 βY35F and increases in βY35A. 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 βY35F or βY35A. X‐ray crystal structures of deoxy βY35F and βY35A are highly isomorphous with the structure of wild‐type deoxyhemoglobin. The βY35F mutation repositions the carboxyl group of Asp126α1 so that it may form a more favorable interaction with the guanidinium group of Arg141α2. The βY35A mutation results in increased mobility of the Arg141α side chain, implying that the interactions between Asp126α1 and Arg141α2 are weakened. Therefore, the changes in the functional properties of these 35β mutants appear to correlate with subtle structural differences at the C terminus of the α‐subunit.

Probing the Conformation of Hemoglobin Presbyterian in the R-State

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(β)->Lys mutation–induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the α1β1 and α1β2 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(α) of Tg-HbP, a residue of the α1β1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the α1β2 interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(β). In short, Lys-108(β) in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbAs affinity to oxygen.

Modeling inhibitors in the active site of aspartate aminotransferase

The rational design of drugs and other inhibitors is a major goal of contemporary enzymology. Among the target enzymes are brain 4-aminobutyrate aminotransferase and insect glutamate decarboxylase. The three-dimensional structures of these enzymes are not yet known, but that of the related aspartate aminotransferases is known.’-6 Like the other two enzymes, aspartate aminotransferase depends upon the coenzyme pyridoxal 5’-phosphate (PLP) and acts on the substrate L-glutamate. We are working with the cytosolic enzyme from pig hearts. Not only is its structure known to high resolution in two conformations, but a large number of both competitive and enzyme-activated inhibitors have been investigated.’-“ It is, therefore, appropriate to attempt rational design of inhibitors for this enzyme. Domain movement plays a critical role in the mechanism of action of aspartate aminotransferase. The enzyme exists in an “open” conformation able to bind substrates or to release products (FIG. 1) and a “closed” conformation in which the protein has folded around a substrate or inhibitor (FIG. 2). This conformational change, which is triggered by substrate binding, involves a domain movement that brings several nonpolar side chains, a “hydrophobic plug,” over the substrate binding cavity. For the pig cytosolic isoenzyme, these hydrophobic residues are V17, F18,121, and V37. (The standard one-letter abbreviations will be used for amino acids.) The substrate binding site includes two critical arginine residues, R386 and R292* (the * denotes that R292 is from the neighboring subunit), that interact with the substrate a-carboxyl and side-chain carboxyl groups, respectively. In the absence of substrate, R386 and R292* are accessible to solvent in the open conformation but not in the closed conformation. Therefore, despite the fact that the hydrophobic side chains of V17, F18,121, and V37 also have a great deal of solvent-accessible surface in the open conformation, on balance the closed conformation is observed to be thermodynamically less stable in the absence of substrate because R386 (and to a lesser degree R292*) will be buried in this conformation without access to polar or charged residues that would compensate for

[80] Investigation of crystalline enzyme—Substrate complexes of pyridoxal phosphate-dependent enzymes☆

Publisher Summary Many pyridoxal phosphate (pyridoxal-P) dependent enzymes have been crystallized, but few of the crystals have been studied either by X-ray crystallography or by other physical techniques. Recently, three groups have initiated crystallographic studies on aspartate aminotransferases. The cytosolic enzyme from chicken hearts and from pig hearts has been prepared in orthorhombic forms, while the mitochondrial isoenzyme of chicken heart has been crystallized in a triclinic form. Many pyridoxal-P enzymes form very small and relatively insoluble crystals. However, if the solubility of these enzymes is increased by changing the pH or ionic composition of the buffer, it may be possible to use the polyethyleneglycol method to obtain larger crystals. This chapter describes the preparation of crystals of the cytosolic isoenzyme of aspartate aminotransferase from pig heart and crystals of enzyme-substrate or enzyme-inhibitor complexes. Crystals of enzyme containing analogs of pyridoxal-P can be prepared by reconstituting the apoenzyme and then treating it as above for the native enzyme.