The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein.

BACKGROUND: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition. RESULTS: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 A resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel alpha/beta fold and contains a Zn2+ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (alpha1(*)-loop12-alpha2(*); where * represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23-alpha3(*)-loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7. CONCLUSIONS: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala.

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.

Induction of an electrogenic transfer of monovalent cations (K+, NH4+) in thylakoid membranes by N,N'-dicyclohexylcarbodiimide.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on photosynthetic electron transport and light-induced NH4+ and K+ uptake in the presence of ammonium or nigericin was studied. DCCD alone had no effect on either the electron transport or the uptake of protons. The simultaneous action of DCCD and low concentrations of ammonium or nigericin was shown to lead to a significant increase in the electron transport rate and a decrease in the steady-state uptake value of H+ and NH4+ or K+. The effect of DCCD on these processes was compared with the effect of the ionophore, valinomycin, which transports potassium and ammonium cations through membranes. The conclusion was made that 1.0-1.5 mol DCCD per mol chlorophyll activated the transfer system of monovalent cations (K+ and NH4+) in thylakoid membranes.

[Anesthesiologic protection of the mother and fetus during abdominal delivery]. / Anesteziologicheskaia zashchita organizma materi i ploda pri abdominal'nom rodorazreshenii.

Central hemodynamics at different stages of anesthesia, changes in hexenal concentration in the mother's body, adequacy of anesthesia and hexenal effect on the condition of the fetus and newborn have been studied in 40 pregnant women without any accompanying extragenital pathology subject to planned cesarean section performed under combined general anesthesia. Normodynamic type of circulation has been established. The analysis of blood catecholamine changes confirmed the adequacy of the anesthesia. The studies of hexenal concentration have demonstrated the absence of negative effect on the newborn delivered by cesarean section.

[Mechanisms of contractile action of acetylcholine on hepatic veins].

In acute experiments on anesthetized rats, acetylcholine (Ach) constricts hepatic venous vessels, causing blood mobilization from the liver, and dilates the sphincters of hepatic veins at the exit from this organ, contributing to the intensification of the outflow of blood deposited in the liver. Vasoconstrictor reactions of capacitive vessels of the liver to Ach are realized through M-cholinoreceptors on endotheliocytes with further involvement of messenger, possibly noradrenaline, which activates alpha-adrenoreceptors on smooth muscle cells (SMC) of capasitive vessels. Dilation of Hv sphincters is carried out due to Ach-induced release of messenger in the vessel wall, probably adrenaline, which in turn activates beta-adrenoreceptors on SMC of the Hv. It is possible, that in such reaction partially involved NO.

Vasoconstrictor effect of acetylcholine in veins of the liver.

Intraportal acetylcholine administered to narcotized rats produced atropine-resistant constriction of hepatic veins, which was considerably prevented by phentolamine. Sodium nitroprusside produced a vasodilator effect. Similar results were obtained on isolated venous strip from the portal vein: acetylcholine-induced contraction was reduced by 25-50% in the presence of nicotinic receptor antagonist tubocurarine and cholinergic agonist nicotine and by 10% in the presence of tetrodotoxin. Probably, acetylcholine stimulates synthesis and release of a vasoconstrictor transmitter via nicotinic receptors of endothelial cells and/or portal vascular wall nerve terminals.

[Elucidation of the mechanisms of acetylcholine constrictor action on the portal vein and its intrahepatic branches].

Intraportal administration of acetylcholine (Ach) to anesthetized rats evokes endothelium depended, atropine resistant and phentholamine sensitive constriction of hepatic portal vessels. On the contrary to Ach action sodium nitroprussideresulted in vasodilatation in this vessel. On the isolated segment of portal vein (PV) similar results were obtained; at the same time the blockade of nicotinic acetylcholinic receptors by nicotine (in high concentration), tubocurarine or tetrodotoxine diminished constrictor reactions of PV to Ach. We concluded that described vasomotor effects of Ach in the hepatic portal bed are carried out through nicotinic Ach-receptors localized on endothelial cells and (or) adrenergic neurones in the wall of portal vessels. These cells synthesize and release mediators, possibly, noradrenaline which causes constriction of portal vessels.

Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures.

The three-dimensional structure of canavalin from jack bean (Canavalia ensiformis).

The three-dimensional structure of the vicilin storage protein canavalin, from Canavalia ensiformis, has been determined in a hexagonal crystal by x-ray diffraction methods. The model has been refined at 2.6 A resolution to an R factor of 0.197 with acceptable geometry. Because of proteolysis, 58 of 419 amino acids of the canavalin polypeptide are not visible in the electron density map. The canavalin subunit is composed of two extremely similar structural domains that reflect the tandem duplication observed in the cDNA and in the amino acid sequence. Each domain consists of two elements, a compact, eight-stranded beta-barrel having the "Swiss roll" topology and an extended loop containing several short alpha-helices. The root mean square deviation between 84 pairs of corresponding C alpha atoms making up the strands of the two beta-barrels in a subunit is 0.78 A, and for 112 pairs of structurally equivalent C alpha atoms of the two domains the deviation is 1.37 A. The interface between domains arises from the apposition of broad hydrophobic surfaces formed by side chains originating from one side of the beta-barrels, supplemented by at least four salt bridges. The interfaces between subunits in the trimer are supplied by the extended loop elements. These interfaces are also composed primarily of hydrophobic residues supplemented by six salt bridges. The canavalin subunits have dimensions about 40 x 40 x 86 A, and the oligomer is a disk-shaped molecule about 88 A in diameter with a thickness of about 40 A. The distribution of domains lends a high degree of pseudo-32-point group symmetry to the molecule. There is a large channel of 18 A diameter, lined predominantly by hydrophilic and charged amino acids, running through the molecule along the 3-fold axis. The majority of residues conserved between domains and among vicilins occur at the interface between subunits but appear otherwise arbitrarily distributed within the subunit, although predominantly on its exterior.

Structures of three crystal forms of the sweet protein thaumatin.

Three crystal forms of the sweet-tasting protein thaumatin from the African berry Thaumatococcus daniellii have been grown. These include two naturally occurring isoforms, A and B, that differ by a single amino acid, and a recombinant form of isoform B expressed in yeast. The crystals are of space groups C2 with a = 117.7, b = 44.9, c = 38.0 A, and beta = 94.0 degrees, P2(1)2(1)2(1) with a = 44.3, b = 63.7 and c = 72.7 A, and a tetragonal form P4(1)2(1)2 with a = b = 58.6 and c = 151.8 A. The structures of all three crystals have been solved by molecular replacement and subsequently refined to R factors of 0.184 for the monoclinic at 2.6 A, 0.165 for the orthorhombic at 1.75 A, and 0.181 for the tetragonal, also at 1.75 A resolution. No solvent was included in the monoclinic crystal while 123 and 105 water molecules were included in the higher resolution orthorhombic and tetragonal structures, respectively. A bound tartrate molecule was also clearly visible in the tetragonal structure. The r.m.s. deviations between molecular structures in the three crystals range from 0.6 to 0.7 A for Calpha atoms, and 1.1 to 1.3 A for all atoms. This is comparable to the r.m.s. deviation between the three structures and the starting model. Nevertheless, several peptide loops show particularly large variations from the initial model.

Structure of a ribonuclease B+d(pA)4 complex.

The structure of a tetragonal crystal of bovine pancreatic RNase B complexed with d(pA)(4) was determined by molecular replacement and difference Fourier methods. This crystal belongs to space group P4(1)2(1)2 and has unit-cell dimensions a = b = 44.5, c = 156.5 A. The model consists of the enzyme and a tetranucleotide with fractional occupancies, suggesting multiple modes of oligonucleotide binding. It does not include any polysaccharide residues or solvent molecules. After refinement at 2.7 A, the R value was 0.163 with acceptable stereochemistry. The model illustrates a set of well defined interactions for substrate binding, particularly between the central dinucleotide and the enzyme.

The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 A resolution.

The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P2(1)3 and P6(3), respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary-structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins.

Determination of three crystal structures of canavalin by molecular replacement.

Canavalin, the major reserve protein of the jack bean, was obtained in four different crystal forms. From the structure determined by multiple isomorphous replacement in a hexagonal unit cell, the structures of three other crystals were determined by molecular replacement. In two cases, the rhombohedral and cubic crystals, placement was facilitated by coincidence of threefold molecular symmetry with crystallographic operators. In the orthorhombic crystal the canavalin trimer was the asymmetric unit. The rhombohedral, orthorhombic and cubic crystal structures were subsequently refined using a combination of several approaches with resulting R factors of 0.194, 0.185 and 0.211 at resolutions of 2.6, 2.6 and 2.3 A, respectively. Variation in the conformation of the molecule from crystal to crystal was small with an r.m.s. deviation in Calpha positions of 0.89 A. Packing is quite different among crystal forms but lattice interactions appear to play little role in the conformation of the molecule. Greatest variations in mean position are for those residues that also exhibit the greatest thermal motion. Crystal contacts in all crystals are mediated almost exclusively by hydrophilic side chains, and three to six intermolecular salt bridges per protein subunit are present in each case.

The crystallographic structure of the subtilisin protease from Penicillium cyclopium.

The major extracellular protease from the fungus Pencillium cyclopium was crystallized in the presence of p-phenylmethanesulfonyl fluoride (PMSF) and investigated by X-ray diffraction analysis. It was subsequently cloned and the amino acid sequence deduced from its cDNA. Although the sequence is only 49% identical to that of proteinase K of Tritirachium album, the three-dimensional structures of the two proteases are virtually identical. The model for P. cyclopium protease was refined by simulated annealing to an R of 18% at 1.7 A resolution. The greatest variation from the proteinase K polypeptide is in loop 114-134 and is due to the absence of a disulfide bridge in the P. cyclopium protease that is present in proteinase K. A difference was also observed in the orientation of the histidine in the catalytic triad, though this could be due to the presence of PMSF at the active site. The coordination geometry of the strongly bound calcium in the P. cyclopium protease is octahedral and uses some different protein ligands than does proteinase K. In the protease from P. cyclopium there is no cysteine near the active site, nor is there a second calcium binding site as is found in proteinase K, suggesting that neither is important to catalytic activity.

Structure of orthorhombic crystals of beef liver catalase.

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the <001> direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the 'bottoms' and 'tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM.

Crystallization and preliminary crystallographic analysis of the Escherichia coli tyrosine aminotransferase.

Tyrosine aminotransferase catalyzes transamination for both dicarboxylic and aromatic amino-acid substrates. The substrate-free Escherichia coli tyrosine aminotransferase (eTAT) bound with the cofactor pyridoxal 5'-phosphate (PLP) was crystallized in the trigonal space group P3(2). A low-resolution crystal structure of eTAT was determined by molecular-replacement methods. The overall folding of eTAT resembles that of the aspartate aminotransferases, with the two identical subunits forming a dimer in which each monomer binds a PLP molecule via a covalent bond linked to the epsilon-NH(2) group of Lys258. Comparison of the structure of eTAT with those of the open, half-open or closed form of chicken or E. coli aspartate aminotransferases shows the eTAT structure to be in the open conformation.