共查询到20条相似文献,搜索用时 31 毫秒
1.
D B Wigley S J Gamblin J P Turkenburg E J Dodson K Piontek H Muirhead J J Holbrook 《Journal of molecular biology》1992,223(1):317-335
We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate). 相似文献
2.
A second symmetry mismatch at the portal vertex of bacteriophage T7: 8-fold symmetry in the procapsid core 总被引:2,自引:0,他引:2
Cerritelli ME Trus BL Smith CS Cheng N Conway JF Steven AC 《Journal of molecular biology》2003,327(1):1-6
Like other bacteriophages, T7 has a singular vertex that is the site of a symmetry mismatch involving the portal/connector protein, a 12-fold ring at the vertex site which is also a 5-fold axis for the icosahedral capsid. In the mature virion, a 6-fold-symmetric tail extends outwards from the connector. T7 also has a cylindrical "core" that assembles on the inner surface of the connector during procapsid formation, is retained in the mature virion, and is required for infectivity. We have investigated the core structure by cryo-electron microscopy and image analysis of procapsids and find that it observes 8-fold symmetry. Stoichiometry data indicate that its major constituent is an octamer of gp15. 相似文献
3.
The structure of the protein subunit of satellite tobacco necrosis virus has been solved at 3.7 Å resolution. We have now crystallographically refined the original model and extended the resolution to 2.5 Å in order to get a model accurate enough to explain the details of the subunit interactions. The refinement was done with a novel method utilizing the icosahedral symmetry of the virus particle.The final model shows a complicated network of interactions, involving salt linkages, hydrogen bonds and hydrophobic contacts. In addition, we have located three different metal ion sites in the protein shell, linking the protein subunits together. These sites are probably occupied by calcium ions. One site is found in a general position near the icosahedral 3-fold axis of the virus. The ligands form an octahedral arrangement, with two main chain carbonyl oxygens (O-61 and O-64), one carboxylate oxygen (OD1 from Asp194) of the same subunit and a second carboxylate oxygen (OE1 of Glu25) from a 3-fold related subunit. Two water molecules complete the octahedral arrangement. A second site is on the icosahedral 3-fold axis and is liganded by the carboxylate oxygens of the 3-fold related Asp55 residues. The third metal ion site is found on the 5-fold axis, liganded by the five carbonyl oxygens of Thr138 and two water molecules.We are unable to locate the first 11 N-terminal amino acid residues, which point into the virus interior. No interpretable density for RNA has been found, indicating that the nucleic acid of the virus does not have a unique orientation in the crystal. 相似文献
4.
Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate. The enzyme appears to be responsible for repairing the unique protein-DNA linkage that occurs when eukaryotic topoisomerase I becomes stalled on the DNA in the cell. The 1.69 A crystal structure reveals that human Tdp1 is a monomer composed of two similar domains that are related by a pseudo-2-fold axis of symmetry. Each domain contributes conserved histidine, lysine, and asparagine residues to form a single active site. The structure of Tdp1 confirms that the protein has many similarities to the members of the phospholipase D (PLD) superfamily and indicates a similar catalytic mechanism. The structure also suggests how the unusual protein-DNA substrate binds and provides insights about the nature of the substrate in vivo. 相似文献
5.
The structure of tumor necrosis factor-alpha at 2.6 A resolution. Implications for receptor binding 总被引:19,自引:0,他引:19
The three-dimensional structure of tumor necrosis factor (TNF-alpha), a protein hormone secreted by macrophages, has been determined at 2.6 A resolution by x-ray crystallography. Phases were determined by multiple isomorphous replacement using data collected from five heavy atom derivatives. The multiple isomorphous replacement phases were further improved by real space symmetry averaging, exploiting the noncrystallographic 3-fold symmetry of the TNF-alpha trimer. An atomic model corresponding to the known amino acid sequence of TNF-alpha was readily built into the electron density map calculated with these improved phases. The 17,350-dalton monomer forms an elongated, antiparallel beta-pleated sheet sandwich with a "jelly-roll" topology. Three monomers associate intimately about a 3-fold axis of symmetry to form a compact bell-shaped trimer. Examination of the model and comparison to known protein structures reveals striking structural homology to several viral coat proteins, particularly satellite tobacco necrosis virus. Locations of residues conserved between TNF-alpha and lymphotoxin (TNF-beta, a related cytokine known to bind to the same receptors as TNF-alpha) suggest that lymphotoxin, like TNF-alpha, binds to the receptor as a trimer and that the general site of interaction with the receptor is at the "base" of the trimer. 相似文献
6.
Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild-type enzyme. The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I. 相似文献
7.
The F43W and F43W/H64L myoglobin (Mb) mutants have been constructed to investigate effects of an electron rich oxidizable amino acid residue in the heme vicinity on oxidation activities of Mb. The Phe-43 --> Trp mutation increases the rate of one-electron oxidation of guaiacol by 3-4-fold; however, the peroxidase activity for F43W/H64L Mb is less than that of the F43W single mutant because the absence of histidine, a general acid-base catalyst, in the distal heme pocket suppresses compound I formation. More than 15-fold improvement versus wild-type Mb in the two-electron oxidation of thioanisole and styrene is observed with the Phe-43 --> Trp mutation. Our results indicate that Trp-43 in the mutants enhances both one- and two-electron oxidation activities (i.e., F43W Mb > wild-type Mb and F43W/H64L Mb > H64L Mb). The level of (18)O incorporation from H2(18)O2 into the epoxide product for the wild type is 31%; however, the values for F43W and F43W/H64L Mb are 75 and 73%, respectively. Thus, Trp-43 in the mutants does not appear to be utilized as a major protein radical site to form a peroxy protein radical in the oxygenation. The enhanced peroxygenase activity might be explained by the increase in the reactivity of compound I. However, the oxidative modification of F43W/H64L Mb in compound I formation with mCPBA prevents us from determining the actual reactivity of the catalytic species for the intact protein. The Lys-C achromobacter digestion of the modified F43W/H64L mutant followed by FPLC and mass analysis shows that the Trp-43-Lys-47 fragment gains a mass by 30 Da, which could correspond two oxygen atoms and loss of two protons. 相似文献
8.
Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis. 相似文献
9.
Purification and characterization of the adenosine A2-like binding site from human placental membrane 总被引:1,自引:0,他引:1
We have purified and characterized the adenosine A2-like binding site from human placental membranes. 5'-N-Ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binds to this site, with a Kd of 240 nM and a Bmax of 13.0 pmol/mg in human placental membranes. The adenosine A2-like binding site was purified after extraction from placental membranes with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The purification included ammonium sulfate precipitation and concanavalin A, DEAE-Sephadex, and Sepharose 6B gel filtration chromatographies. The protein was purified 127-fold to homogeneity, with a final specific activity of 1.5-1.9 nmol/mg of protein and a 5.5-8.1% yield of binding activity from the membranes. The purified protein had similar binding properties and an identical potency order for displacement of [3H] NECA by adenosine analogs as the initial membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa which coeluted with [3H]NECA binding activity during Sepharose 6B gel filtration chromatography. In 0.1% Triton X-100, the binding complex has a Stokes radius of 70 A, a sedimentation coefficient of 6.9 S, and a partial specific volume of 0.698 ml/g. The detergent-protein complex has a calculated molecular mass of 230 kDa. The estimated frictional ratio is 1.5. The native binding complex appears to consist of a dimer of identical subunits. The function of this ubiquitous protein remains unclear. 相似文献
10.
H A Tajmir-Riahi 《Biophysical chemistry》1986,25(1):37-41
Interaction between [Co(NH3)5Cl]Cl2, [Co(NH3)4Cl2]Cl and L-ascorbic acid has been investigated in aqueous solution and solid complexes of the type [Co(NH3)5 ascorbate]Cl2 X H2O and [Co(NH3)4 ascorbate]Cl2 X H2O have been isolated and characterized by 13C-NMR, FT-IR and electron absorption spectroscopy. Spectroscopic and other evidence suggested that the sugar anion binds monodentately in the [Co(NH3)5 ascorbate]2+ cation via the ionized O3 oxygen atom and bidentately in [Co(NH3)4 ascorbate]2+ through the O1 and O4 oxygen atoms, resulting in a six-coordinate geometry around the Co(III) ion. The intermolecular sugar hydrogen-bonding network is perturbed upon sugar metalation and the sugar moiety shows a similar conformation to that of the sodium ascorbate compound in these series of cobalt-ammine complexes. 相似文献
11.
Tsitsanou KE Skamnaki VT Oikonomakos NG 《Archives of biochemistry and biophysics》2000,384(2):245-254
Caffeine, an allosteric inhibitor of glycogen phosphorylase a (GPa), has been shown to act synergistically with the potential antidiabetic drug (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5,6-tricarboxylate (W1807). The structure of GPa complexed with caffeine and W1807 has been determined at 100K to 2.3 A resolution, and refined to a crystallographic R value of 0.210 (Rfree = 0.257). The complex structure provides a rationale to understand the structural basis of the synergistic inhibition between W1807 and caffeine. W1807 binds tightly at the allosteric site, and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface which transform GPa to the T'-like state conformation already observed with GPa-glucose-W1807 complex. A disordering of the N-terminal tail occurs, while the loop of polypeptide chain containing residues 192-196 and residues 43'-49', from the symmetry related subunit, shift to accommodate W1807. Caffeine binds at the purine inhibitor site by intercalating between the two aromatic rings of Phe285 and Tyr613 and stabilises the location of the 280s loop in the T state conformation. 相似文献
12.
Satellite tobacco necrosis virus (STNV) is an icosahedral virus which contains three classes of Ca2+ binding site. One of these classes, a five-fold carbonyl site which is believed to exist in a Ca2+ channel, has been investigated using laser-induced Eu3+ luminescence spectroscopy. These twelve identical sites are rather rigid, as evidenced by the single narrow (full width at half-maximum is 6.5 cm-1) band observed at 579.58 nm in the 7F0----5D0 excitation spectrum of the Eu(3+)-STNV complex. Lifetimes of 270 microseconds in H2O and 1620 microseconds in D2O indicate that there are three water molecules bound to the Eu3+ at this site. Ligand field splitting of the 7F0----5D1 and 7F0----5D2 excitation spectra show that this site possesses fairly high symmetry (less than or equal to C5V). The Eu3+ complex of nitrilotriacetic acid was determined via titration to have a dissociation constant, Kd, of 20 +/- 2 nM; this value has been used in competition experiments to deduce that the virus site class binds Eu3+ with a Kd of 1.1 +/- 0.3 nM. This putative ion channel demonstrates remarkable size selectivity, with lanthanide affinities varying by more than one order of magnitude. 相似文献
13.
Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. However, the concentration of RBP in the plasma is limiting, and the complex isolated from serum is composed of TTR and RBP in a 1 to 1 stoichiometry. We report here the crystallographic structure at 3.2 A of the protein-protein complex of human RBP and TTR. RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry: Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR. 相似文献
14.
M E Cuff W A Hendrickson J Lamy J N Lamy K I Miller K E van Holde 《Journal of molecular biology》1990,213(1):11-15
The carboxyl-terminal oxygen-binding unit of the polypeptide from Octopus dofleini hemocyanin has been crystallized in a form suitable for three-dimensional X-ray analysis. This proteolytic fragment has a molecular weight of 47 kDa and reversibly binds O2 while exhibiting a slight Bohr effect. Two types of crystals have been grown. Type I crystals, currently under analysis, belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of 92.6 A x 167.4 A x 59.2 A. A composition of two protein molecules per asymmetric unit and 50% solvent content is consistent with a self-rotation function that identifies a non-crystallographic 2-fold axis of symmetry relating these molecules. Diffraction extending beyond 1.9 A Bragg spacings can be detected with synchrotron X-radiation. 相似文献
15.
F Kesbeke J Baraniak R Bulgakov B Jastorff M Morr G Petridis W J Stec F Seela P J Van Haastert 《European journal of biochemistry》1985,151(1):179-186
The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2'H. Modifications at N7, C8, O3' and O5' induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold. Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3', while modifications at N2H2, N3, N7, O2'H, and O5' have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein. cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3-6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site. 相似文献
16.
X-ray analysis of the disk of tobacco mosaic virus protein. 3. A low resolution electron density map
The structure of the disk of tobacco mosaic virus protein at low resolution has been determined by X-ray crystal analysis. Signs for the three principal projections were found by isomorphous replacement, using a mercury derivative. The heavy-atom positions were located by interpretation of difference Patterson maps on the basis of the non-crystallographic 17-fold rotational symmetry of the disk, and of the packing of the disks determined in the preceding paper (Finch et al., 1974).The electron density in the corresponding three projections was computed to a resolution of 6 Å. From the projections in the [100] and [010] directions, which are at right-angles to the 17-fold rotation axis, a three-dimensional electron density map has been calculated making use of the non-crystallographic symmetry. The procedure is similar to the three-dimensional image reconstruction technique used in electron microscopy.The map indicates that the subunits in the two rings face the same way and, hence, that the disk is polar. There are differences between the subunits in the two rings at high radius, which are presumably a consequence of the pairing interaction responsible for stabilizing the two-layer polar structure. The map has been compared with a low-resolution map of the intact virus, and certain common features can be identified, notably the site of the nucleic acid. 相似文献
17.
18.
Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. 相似文献
19.
Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate 总被引:5,自引:0,他引:5
The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
20.
Structural basis of the ribosomal machinery for peptide bond formation,translocation, and nascent chain progression 总被引:9,自引:0,他引:9
Bashan A Agmon I Zarivach R Schluenzen F Harms J Berisio R Bartels H Franceschi F Auerbach T Hansen HA Kossoy E Kessler M Yonath A 《Molecular cell》2003,11(1):91-102
Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism. 相似文献