Low resolution crystal structure of hagfish insulin

Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P4₁2₁2 with a monomer in the asymmetric unit. The application of the Rossmann &; Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 Å resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.     

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.     

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.     

500-MHz1H NMR studies of insulin: Complete assignment of histidine resonances

The two histidines of the insulin monomer play a vital role in the organization of insulin into insulin hexamers. The B10 histidines bind to zinc to form two-zinc insulin hexamer, and both the B5 and B10 histidines are implicated in the formation of four-zinc insulin hexamer. These two histidines are both accessible to solvent in the dimeric form of insulin, the predominant species present at pH 2–3. In the present work we report the first 500-MHz¹H NMR studies of insulin. At this frequency all four proton resonances from the two histidines of each equivalent monomer are resolved. The resonances are assigned to the C(2)- and C(4)-imidazole protons of B5 His and B10 His employing Carr-Purcell pulse sequences to detect singlets and to observe approximateT ₂ relaxation times. Zinc-free bovine insulin at pH 2.9 was examined at temperatures up to 60°C in acetate buffer and in urea of varying concentrations. The environments of B5 His in molecule I and molecule II of the dimer must be the same, with the same being true for B10 His, since a total of only four sharp resonances are seen. Our assignments for the two C(2) protons are consistent with those determined from recent studies of human (B5 Ala) insulin.     

Ferriprotoporphyrin IX Oxygen Activation/Insertion Reactions: The Nature of the Interaction between Ferriprotoporphyrin IX and Aniline (Substrate)

The interaction between aniline and ferriprotoporphyrin IX in alkaline solution has been investigated using pyridine and the N-methyl pyridinium ion as site-specific inhibitors of oxygen activation. Pyridine inhibits oxygen activation in a noncompetitive manner with respect to aniline (K₁ = 1.24 mol ⁻¹ dm³ at 30°C) while the N-methyl pyridinium ion inhibited in a manner consistent with two sites for aniline binding, only one of which was competitively inhibited (K₁ = 317mol^-l dm³ at 30°C). A comprehensive reinvestigation of the interaction of pyridine and N-methyl pyridinium ions with alkaline ferriprotoporphyrin IX has shown that two molecules of each ligand bind per hemin dimer in a strongly cooperative manner. The association constant for the first pyridinium ion bound is K_1a = 176 mol⁻¹ dm³ at 30°C, while that for the first pyridine molecule bound is K_1a = 0.580 mol⁻¹ dm³ at 30°C; these are both close to the observed inhibition association constants (K₁). Thermodynamic parameters for the interactions have been evaluated and compared to previous literature values. On the basis of these results a model is proposed for aniline interaction with the ferriprotoporphyrin dimer IX which involves the binding of two molecules of aniline to the ferriprotoporphyrin IX tetrapyrrole ring system by planar π bonding interactions with the rings having the propionate groups attached.     

Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase,as observed in a new crystal form: Implications for the reaction mechanism

The structure of trypanosomal triosephosphate isomerase (TIM)has been solved at a resolution of 2.1Å in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 Å, 48.3 Å, 131.0 Å, 90.0°, 100.3°, 90.0°), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the “Open” conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 Å resolution), and with 40 μM phosphoglycolohydroxamate (studied at 2.5 Å resolution). It is found that transfer to 0.4 M ammonuum sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a “closed” conformation. In a mother liquor containing 40 μM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding. © 1993 Wiley-Liss, Inc.     

Ventilation and oxygen consumption in the hagfish, Myxine glutinosa L.

Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min^?, and 18.2 ± 5.1·min^?, respectively, at 7°C to 1.70 ± 0.20 ml·min^?, and 70.1 ± 9.5·min^? at 15 ·C.Standard oxygen consumption,V?O₂, was measured in non-buried hagfish. V?O₂ was 0.57 ± 0.17μl O₂·g^?1·min^?1 at 7°C, and 0.85 ± 0.12μl O₂·g^?1·min^?1 at 15°C.     

Crystallographic refinement and atomic models of two different forms of citrate synthase at 2·7 and 1·7 Å resolution

The structures of pig heart and chicken heart citrate synthase have been determined by multiple isomorphous replacement and restrained crystallographic refinement for two crystal forms, a tetragonal form at 2·7 Å and a monoclinic form at 1·7 Å resolution, with crystallographic R-values of 0·199 and 0·192, respectively. The structure determination involved a novel application of restrained crystallographic refinement, in that the refinement of incomplete models was necessary in order to completely determine the course of the polypeptide chain. The recently determined amino acid sequence (Bloxham et al., 1981) has been fitted to the models. The molecule has substantially different conformations in the two crystal forms, and there is evidence that a conformational change is required for enzymatic activity.The molecule is a dimer of identical subunits with 437 amino acid residues each. The conformation is all α-helix, with 40 helices per dimer packing tightly to form a globular molecule. Many of the helices are kinked in various ways or bent smoothly over a large angle. Several of the helices show an unusual antiparallel packing.Each subunit is clearly divided into a large and a small domain. The two crystal forms differ by the relative arrangement of the two domains. The tetragonal form represents an open configuration with a deep cleft between the two domains, the monoclinic form is closed. The structural change from the open to the closed form can be described by an 18 ° rotation of the small domain relative to the large domain.Crystallographic analyses were performed with the product citrate bound in both crystal forms, with coenzyme A (CoA) and a citryl-CoA analogue bound to the monoclinic form. These studies establish the CoA and the citrate binding sites, and the conformations of the two product molecules in atomic detail. The subunits are extensively interdigitated, with one subunit making significant contributions to both the citrate and the CoA binding sites of the other subunit. The adenine moiety of CoA is bound to the small domain, and the pantothenic arm is bound to the large domain. The citrate molecule is bound in a cleft between the large domain. The citrate molecule is bound in a cleft between the large and small domain, with the si carboxymethylene group facing the SH arm of coenzyme A. In the monoclinic form, the cysteamine part of CoA shields the bound citrate completely from the solution. Partial reaction of CoA-SH and aspartate 375 to form aspartyl-CoA, and citrate to form citryl-CoA may occur in the crystals. The conformation of CoA is compact, characterized by an internal hydrogen bond O-52 … N-7 and a tightlybound water molecule O-51 … HOH … O-20.     

Porcine superoxide dismutase

Summary A cuprozinc superoxide dismutase has been isolated from pig liver. The enzyme is similar to previously described cuprozinc superoxide dismutases in that it is a dimer of about 32 000 molecular weight consisting of approximately two equally sized subunits, and 2 atoms of copper and two atoms of zinc per molecule. It differs, however, from previously described cuprozinc superoxide dismutases because of its higher isoelectric point; pI 6.8 vs 4.9 for bovine enzyme. The diffusion coefficient for the porcine enzyme was determined to be 7.53×10⁻⁷ cm²s⁻¹, while the equivalent spherical hydrodynamic radius was computed as 28.5 ?. The enzyme was observed to undergo self-association with time. Sulfhydryl interaction is postulated to be involved.     

Crystal structures of deoxy- and carbonmonoxyhemoglobin F1 from the hagfish Eptatretus burgeri

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 A, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the alpha1beta1 interface of all vertebrate alpha2beta2 tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha2beta2 tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.     

Structural relationships in the two-zinc insulin hexamer.

The refinement of the crystal structure of two-Zn pig insulin using 1.5-A (1A = 0.1 nm) resolution data by Fourier and fast Fourier least-squares methods allows us to make detailed comparisons between the two independent molecules present in the two-Zn insulin dimer and to describe their interactions in the monomer, dimer, and hexamer. The main chain structures for the two molecules agree well except at the N terminus of the A chain and the C terminus of the B chain. The residues along the line of the local two-fold axes, apart from the B25 side chain, conform extremely closely to the two-fold symmetry, although the discrepancies are much more apparent away from this axis. The ability of the insulin molecule to adopt different conformations may be an important factor in the expression of its biological activity.     

Behaviour of radioactive palmitic acid during heat treatment of bovine serum albumin

A radioactive complex AP_2.3 (A: bovine serum albumin, P: radioactive palmitic acid) has been prepared and incubated at pH 9 and 65°C for 60 min. Analysis by disc gel electrophoresis revealed three zones: zone 1, undenatured monomer; zone 1′, modified monomer; and zone 2, dimer. Counting of sliced gels indicated that only zone 1 was radioactive, meaning that fatty acids are released in the process 1→1′, but not in the process 1′+1′→2. In other words, fatty acids are released from albumin when native albumin is unfolded to form component 1′ during the incubation. The fatty acids released are concentrated on a particular species of albumin molecule which is not changed to component 1′. The percentage of component 1 (p) was 37%. This value agrees with that calculated by the equation which was proposed by us in a previous paper, p = (v/6) × 100%. In the present case v is 2.3.     

Binding of D-glucose to insulin.

Binding of D-glucose to insulin has been studied by equilibrium dialysis. The binding is not very specific and probably takes place in two steps. The average amount of glucose molecules bound per insulin molecule is eight, two molecules in the first and six during the second step of binding. The intrinsic binding constants for both steps are almost the same (6-10-2 M-minus 1 and 1-10-3 M-minus 1) which can be explained by assuming: (1) that after binding of the first two molecules a conformational change of insulin occurs which facilitates the binding of the next six molecules of D-glucose; or (2) that in the second step of binding the glucose binds to hydrophobic regions which are unmasked by dissociation of the insulin dimer. Using a three-dimensional model of the insulin molecule areas of the protein molecule where binding of glucose can occur were selected. The glucose-binding site very probably involves the area at the insulin surface where most of the invariant and modification-selective residues are present.     

Structure of cycloheptaamylose inclusion-complexes: crystal structure of substituted benzoic acid and phenol derivatives

Cycloheptaamylose has been crystallized with 2,5-diiodobenzoic acid as guest. The X-ray crystal structure at 1.2-Å resolution with space group C2 and cell dimensions a  19.192 (13), b  24.759 (20), c  15.739 (13) Å, and β  109.6 (3)° was solved by using rotation-translation functions. Complexes of other meta-substituted guests were found to be isomorphous, and were solved by using the phases of the cycloamylose of the 2,5-diiodobenzoic acid complex. The complex with 2-bromo-5-tert-butylphenol having a  19.235 (11), b  24.662 (17), c  16.018 (11) Å, and β  108.9 (2)° was determined at 1.0 Å resolution, and the complexes with m-bromobenzoic acid, m-iodobenzoic acid, m-iodophenol, m-toluic acid, and 2-bromo-4-tert-butylphenol were determined at 2.0-Å resolution. In all cases, the guest molecule was disordered. However, by using information from all the structures, it may be concluded that the functionally important carboxylic acid group lies in the primary-hydroxyl end of the cycloheptaamylose molecule. As studies in solution have shown that the hydrogen-bonding groups of guest molecules interact with the secondary-hydroxyl end of the cycloheptaamylose molecule, it is concluded that the structure seen in the crystals here does not correspond to a catalytically active species. Cyclo-heptaamylose exists as a dimer in the crystal by means of extensive hydrogen bonding across the secondary-hydroxyl ends of two cycloheptaamylose molecules. A continuous channel throughout the crystal is achieved by the stacking of these dimer units.     

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami: an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83?bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution.     

Structural study of modified fibrinogen microcrystals by electron microscopy

The same molecular shape of proteolytically modified fibrinogen has been identified in two different crystalline forms: orthorhombic P2₁2₁2 with a = 44.7(± 1.7) nm, b = 11.8(± 1.0) nm, c = 3.8 nm and monoclinic P2₁ with a = 17.7(± 0.6) nm, b = 16.2(± 1.0) nm, c = 4.8 nm and β = 95 °. The shape of the molecule is more detailed than has been reported. It has the commonly accepted elongated form 44.5(± 1.5) nm long with a 2-fold axis perpendicular to the major axis of the molecule. Each end domain shows a distinctive substructure and has an asymmetry similar to that reported by Williams (1981) and Mosesson et al. (1981) from single molecule observations. The ends are flattened, having overall dimensions of approximately 12 nm × 9 nm × 4 nm. The major difference between this model and previous models is the absence of a central nodule. There are, however, two small additional protein-dense regions at 5.0 nm from the centre of the rod connecting the two ends. A rough estimate of the relative heights of the different parts of the molecule was obtained using the two different projections of the molecule obtained from the two different crystalline forms. The molecule appears to be slightly bent at the centre, as predicted by Doolittle (1977).     

Morphometics and Gonadal Development of the Hagfish Eptatretus cirrhatus in New Zealand

Hagfishes have been the target of commercial fisheries in many areas of the world, with the catch processed for leather and for human consumption. A fishery has been operating in New Zealand waters for the last six years, harvesting the bearded hagfish, Eptatretus cirrhatus. The fishery has thus far been unregulated. Based on samples collected dockside over a two-year period, this report expands the morphometric database for this species, provides information on the size and weight of the harvested animals, determines the sizes at the onset of gonadal development and the minimum sizes at sexual maturation for males and females, and indicates that E. cirrhatus, like most other hagfish species, has no specific breeding season. Although females appear in the population at smaller sizes, the sex ratio for mature animals is 1:1 and the sizes of the largest males and females are comparable. The changes observed in sex ratio as a function of TL suggest differences in the timing and rates of gonadal development in females versus males rather than protogyny. Based on the size of the eggs, the number of eggs per female, the proportion of the population that contains large eggs, and the number of postovulatory females, it is clear that E. cirrhatus, like other hagfish species, are potentially vulnerable to overexploitation.     

Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.