Structure of a monoclinic crystal from of cyctochrome b1 (Bacterioferritin) from E. coli.

Crystals of E. coli cytochrome b1, alias bacterioferritin, were grown fr om a low ionic strength solution. The resulting monoclniic P21 structure was solved by molecular replacement and refined using noncrystallographi c symmetries applied to the fundamental unit, consisting of two protein subunits and a single haem. From the Patterson self-rotation results it was shown that the asymmetric unit of the monoclinic crystal consists of 12 such dimers and corresponds to a complete, nearly spherical, molecule of bacterioferritin (M4 = 450 kDa) of 432 point-group symmetry. It is thus the most symmetrical cytochrome. As previously determined for the tetragonal form, the haem is located in a special position on a local twofold axis of the dimer. A bimetal centre is also observed within the four-helix bundle of each monomer; a metal-binding site is located on the fourfold axis.

Circumstantial evidence for cytochrome b1 involvement in the functioning of lac-permease in respiring Escherichia coli.

The structure of the haem-binding site of cytochrome b1 and particularly the fact that the two protein ligands of the haem are methionines could explain a correlation found between loss of lac-permease activity and replacement of methionine by norleucine in the protein of aerobically respiring E. coli. If cytochrome b1 is essential for lac-permease mediated transport in whole bacteria as this correlation suggests, translocation of substrate by this permease must be coupled to electron transport. Such a dependence would invalidate the chemiosmotic interpretation of lactose transport in E. coli in its present form and would be in variance with the coupling-by-energy theories of lactose transport that exempted translocation from dependence on energy yielding processes.

Structure solution of a cubic crystal of concanavalin A complexed with methyl alpha-D-glucopyranoside.

The solution of the cubic crystal form (a = 167.8 A) of concanavalin A complexed with the monosaccharide methyl alpha-D-glucopyranoside is described. The space group has been determined as I2(1)3 rather than I23. The use of cadmium to replace cobalt at the transition metal-ion binding site and to replace calcium at its binding site proved to be crucial to the successful solution of the crystal structure. The relatively small isomorphous signals of 21 e(-) for the replacement of cobalt and 28 e(-) for the replacement of calcium, yielded interpretable difference Patterson maps. The electron-density map calculated in space group I2(1)3 at 5.4 A resolution, based on phases derived from single- and double-substituted cadmium differences, revealed a classical concanavalin A tetramer of 222 point symmetry, as seen in all the known crystal structures of concanavalin A. Rigid-body refinement at 3.6 A using the refined coordinates of saccharide-free concanavalin A converged to an R factor of 27.4%. A molecular-replacement analysis, consistent with this crystal structure, and initial experiences in the incorrect space group I23 are described as these also prove to be instructive.

Properties of a crystal of the complex of methyl D-arabinofuranoside with concanavalin A.

The complex of methyl alpha-D-arabinofuranoside with concanavalin A crystallizes in the orthorhombic space group P2(1)22(1) with cell dimensions a = 97.5, b = 87.0 and c = 61.5 A. The asymmetric unit contains one dimer and the unit cell consists of two tetrahedral clusters of point-group symmetry 222. The crystals diffract to 2.0 A resolution.

Refined structure of concanavalin A complexed with methyl alpha-D-mannopyranoside at 2.0 A resolution and comparison with the saccharide-free structure.

The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 A resolution, which was the first detailed study of lectin-saccharide interactions.

A crystallographic study of haem binding to ferritin.

Ferritin, the iron-storage protein, binds porphyrins, metalloporphyrins and the fluorescent dyes ANS (8-anilino-1-naphthalenesulfonic acid) and TNS (2-p-toluidinyl-6-naphthalenesulfonic acid), similarly to apo-myoglobin. Octahedral crystals of horse-spleen apo-ferritin (HSF; 174 amino acids) complexes prepared by the addition of haem, hematoporphyrin or Sn-protoporphyrin IX to a solution of apo-ferritin crystallize in space group F432 with cell parameter a = 184.0 A. X-ray crystallographic analysis of single crystals prepared from a mixture containing haem or Sn-protoporphyrin IX shows that the haem-binding sites in these crystals are occupied by protoporphyrin IX, which is free of metal, rather than by the original metalloporphyrin. The present paper describes the structure of horse-spleen apo-ferritin cocrystallized with Sn-protoporphyrin IX. The 6797 reflections up to 2.6 A resolution used in the refinement were obtained from a data set recorded on a Nicolet/Xentronics area detector with Cu Kalpha radiation from a Rigaku RU 200 rotating anode. The final structure comprises 1613 non-H atoms, two Cd atoms and 170 solvent molecules. Four residues are described as disordered. The root-mean-square deviations from ideal bond lengths and angles are 0.013 A and 2.88 degrees, respectively. Protoporphyrins are observed in special positions on the twofold axes of the ferritin molecule with a stoichiometry of 0.4 per subunit.

High-resolution structures of single-metal-substituted concanavalin A: the Co,Ca-protein at 1.6 A and the Ni,Ca-protein at 2.0 A.

The molecular structures of cobalt- and nickel-substituted concanavalin A have been refined at 1.6 and 2.0 A resolution, respectively. Both metal derivatives crystallize in space group I222 with approximate cell dimensions a = 89, b = 87 and c = 63 A and one monomer in the asymmetric unit. The final R factor for Co-substituted concanavalin A is 17.8% for 29 211 reflections with F > 1.0sigma(F) between 8.0 and 1.6 A. For Ni-substituted concanavalin A the final R factor is 15.9% for 16 128 reflections with F > 1.0sigma(F) between 8.0 and 2.0 A resolution. Both structures contain a transition-metal binding site and a calcium-binding site but, unlike Cd-substituted concanavalin A, do not have a third metal-binding site. The Co-substituted concanavalin A structure diffracts to the highest resolution of any concanavalin A structure reported to date. A comparison of the structures of Ni-, Co-, Cd-substituted and native concanavalin A gives an indication of coordinate errors, which is a useful baseline for comparisons with saccharide complexes of concanavalin A described in other work. We also give a detailed account of multiple conformations which were found for five side-chain residues.

Structure of a unique twofold symmetric haem-binding site.

Bacterioferritin of Escherichia coli, also known as cytochrome b1, is a hollow, nearly spherical shell made up of 24 identical protein subunits and 12 haems. We have solved this structure in a tetragonal crystal form at 2.9 A resolution. We find that each haem is bound in a pocket formed by the interface between a pair of symmetry-related subunits. The quasi-twofold axis of the haem is closely aligned with the local twofold axis relating these subunits. The axial ligands of the haem are sulphurs of two equivalent methionyl residues (Met 52) from the symmetry-related subunits. A cluster of four water molecules is trapped in the gap between the upper edge of the haem and two extended protein loops which close off the haem from the outer aqueous environment. This is the first structure of a bis-methionine ligated haem-binding site and the first case of a twofold symmetric haem-binding site.

Refined structure of cadmium-substituted concanavalin A at 2.0 A resolution.

The three-dimensional structure of cadmium-substituted concanavalin A has been refined using X-PLOR. The R factor on all data between 8 and 2 A is 17.1%. The protein crystallizes in space group I222 with cell dimensions a = 88.7, b = 86.5 and c = 62.5 A and has one protein subunit per asymmetric unit. The final structure contains 237 amino acids, two Cd ions, one Ca ion and 144 water molecules. One Cd ion occupies the transition-metal binding site and the second occupies an additional site, the coordinates of which were first reported by Weinzierl & Kalb [FEBS Lett. (1971), 18, 268-270]. The additional Cd ion is bound with distorted octahedral symmetry and bridges the cleft between the two monomers which form the conventional dimer of concanavalin A. This study provides a detailed analysis of the refined structure of saccharide-free concanavalin A and is the basis for comparison with saccharide complexes reported elsewhere.

Location of haem in bacterioferritin of E. coli.

. A low-resolution partial structure of bacterioferritin was solved using a combination of molecular replacement and rigid-body refinement methods. Modification of bacterioferritin crystals by soaking in tetrachloroplatinate results in a phase transition from tetragonal symmetry (space group P4(2)2(1)2) to a pseudo-cubic one (approximate space group I432). Helical parts of human H ferritin structure stripped of side chains beyond the C(beta) atoms were used as the model. An electron-density map of the refined model revealed a region of extended density which by its shape and position in a pocket between helices was identified as haem. Inclusion of haem in the refinement showed that it can occupy only one of two symmetry-related sites near a twofold axis of the molecule.

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha-D-mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X-ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide-free concanavalin A. The structure of the saccharide complex was refined by restrained least-squares methods to an R-factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha-D-mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium-binding site and 12.8 A from the transition metal-binding site. A network of seven hydrogen bonds connects oxygen atoms O-3, O-4, O-5 and O-6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O-2 and O-1 of the mannoside extend into the solvent. O-2 is hydrogen-bonded through a water molecule to an adjacent asymmetric unit. O-1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms.

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

Chemical and Mössbauer spectroscopic evidence that iron-containing concanavalin A is a ferritin.

We report here the preparation of iron-containing concanavalin A. It has a protein-to-iron ratio of 2.0, and the iron compound it contains is particulate with an average diameter of 85 A. Iron-containing concanavalin A interacts reversibly with dextran and with methyl alpha-D-glucoside. The molecular basis of these findings is discussed and a possible mechanism suggested where one of the molecular forms of concanavalin A has the structure of an apoferritin into which iron is deposited in the form of ferrihydrite.

Properties of a new crystal form of the complex of concanavalin A with methyl alpha-D-glucopyranoside.

The complex of concanavalin A with methyl alpha-D-glucopyranoside crystallizes as regular rhombic dodecahedra containing 35% protein by weight. The crystal is of space group I23 with a = 167.8 A (1 A = 0.1 nm) and contains one concanavalin A dimer per asymmetric unit. It diffracts to a resolution of 1.9 A and is suitable for crystallographic investigation of the structure of the saccharide-binding site.

Preliminary results for the primary structure of bacterioferritin of Escherichia coli.

Bacterioferritins are type-b cytochromes which resemble ferritin. Amino acid analysis combined with chemical modification and partial sequence analysis characterize bacterioferritin of Escherichia coli in terms of its primary structure. It is a protein composed of one kind of polypeptide chain that commences with methionine and terminates with glutamic acid. The length of the polypeptide chain is, tentatively, 146 residues. Besides the N-terminal methionine residue there are three more methionine residues, which yield four CNBr peptides, which have been aligned. The identity of the following positions in the sequence has been ascertained: residues 1-25, 30-37, 83-88, 127-132 and 143-146. No homology with ferritin was found.

Ferritin: design and formation of an iron-storage molecule.

Although essential for most forms of life, too much iron is harmful. To cope with these antagonistic phenomena an iron-storage molecule, ferritin, has evolved. The structure of horse spleen apoferritin, which has recently been refined, consists of 24 symmetrically related subunits forming a near-spherical hollow shell. In ferritin the central cavity is occupied by an iron core of 'ferrihydrite', a geologically ephemeral mineral found in hot or cold springs and in mine workings, or produced in the laboratory by heating solutions of ferric salts. Ferritin itself forms most readily from apoferritin, in the presence of dioxygen, from FeII, not FeIII. Access to its interior is through small intersubunit channels, and the protein influences both the rate of FeII-oxidation and the form of oxide produced.

Dynamics of heme iron in crystals of metmyoglobin and deoxymyoglobin.

The 57Fe gamma-ray resonance absorption spectra have been measured in crystals of metmyoglobin and deoxymyoglobin over a wide range of temperatures. Above a critical temperature common to both proteins (220 K), the dynamics of heme iron display a dramatic change, in that two kinds of thermal fluctuations come into play--a fast fluctuation associated with a steep decrease of the total fluctuation of characteristic time 10(-8) sec, associated with bounded diffusive motion. By using both discrete jump and continuous diffusion models, the latter based on the Brownian motion of an overdamped harmonic oscillator, the essential parameters of the iron motion (mean square displacement and jump frequency or diffusion constant) can be derived as a function of temperature. Thus, for deoxy Mb at 288 K, the mean square displacement for the fast fluctuation is about 6 X 10(-2) A2 and for the diffusive motion is 1.6 X 10(-2) A2; the diffusion constant is 4 X 10(-10) cm2/sec. The diffusive process is associated with an activation energy of about 0.75 kcal/mol. Although the same general kinds of phenomena are observed in crystals of MetMb and deoxy Mb, significant differences in behavior are found, which suggest that the main dynamical phenomenon observed reflects internal large-scale motions of the protein.

The molecular composition of the volutin granule of yeast.

The volutin granule was isolated from yeast by disruption of freeze-dried cells in an organic solvent and density-gradient-gradient centrifugation. The granule is composed of two types of macromolecule, a linear-chain polyphosphate and four basic proteins, of molecular weights ranging from 10 000 to 20 000. In the dissolved granule these macromolecules are in a complex that is uniform by hydrodynamic criteria (s20,w = 22.3 S). The polyphosphate separated from this complex gives a single 31P n.m.r. resonance and in the analytical ultracentrifuge behaves as a monodisperse solute of molecular weight 245 000 +/- 1000. In the 31P n.m.r. spectrum of yeast used for its isolation, this polyphosphate accounts for 14% of total cell polyphosphate.

The composition and the structure of bacterioferritin of Escherichia coli.

Bacterioferritin isolated from Escherichia coli is of two kinds: a protein containing a polynuclear iron compound, the bacterioferritin proper and a protein free of the polynuclear iron compound, the apo-bacterioferritin. Bacterioferritin of both kinds is characterized by absorption maxima at 417,530 and 560 nm, contributed by protohaem IX. Single crystals of bacterioferritin of the space group I432 suggest that the molecule is made up of 24 identical subunits related by a cubic point symmetry. The molecular weight of the protein subunit, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is 15000. In the electron microscope the bacterioferritin molecule appears to be a sphere of 9.5 nm (95 A) diameter composed of a negatively staining outer shell and an inner electron-dense core of 6 nm (60 A) diameter.