Spectroscopic characterization of the electronic changes in the active site of Streptomyces antibioticus tyrosinase upon binding of transition state analogue inhibitors

The dinuclear copper enzyme tyrosinase (Ty) from genetically engineered Streptomyces antibioticus has been investigated in its paramagnetic half-met form [Cu(I)-Cu(II)]. The cw EPR, pulsed EPR, and hyperfine sublevel correlation spectroscopy (HYSCORE) experiments on the half-met-Ty and on its complexes with three different types of competitive inhibitor are reported. The first type includes p-nitrophenol, a very poor substrate for the monooxygenase activity of Ty. The second type comprises hydroxyquinones, such as kojic acid and l-mimosine, and the third type of inhibitor is represented by toluic acid. The electronic and structural differences of the half-met-Ty form induced at the cupric site by the different inhibitors have been determined. Probes of structural effects are the hyperfine coupling constants of the non coordinating Ndelta histidyl nitrogens. By using the available crystal structures of hemocyanin as a template in combination with the spectroscopic results, a structural model for the active site of half-met-Ty is obtained and a model for the binding modes of both mono- and diphenols could be proposed.     

Catalytic mechanism of cyclophilin as observed in molecular dynamics simulations: pathway prediction and reconciliation of X-ray crystallographic and NMR solution data

Cyclophilins are proteins that catalyze X-proline cis-trans interconversion, where X represents any amino acid. Its mechanism of action has been investigated over the past years but still generates discussion, especially because until recently structures of the ligand in the cis and trans conformations for the same system were lacking. X-ray crystallographic structures for the complex cyclophilin A and HIV-1 capsid mutants with ligands in the cis and trans conformations suggest a mechanism where the N-terminal portion of the ligand rotates during the cis-trans isomerization. However, a few years before, a C-terminal rotating ligand was proposed to explain NMR solution data. In the present study we use molecular dynamics (MD) simulations to generate a trans structure starting from the cis structure. From simulations starting from the cis and trans structures obtained through the rotational pathways, the seeming contradiction between the two sets of experimental data could be resolved. The simulated N-terminal rotated trans structure shows good agreement with the equivalent crystal structure and, moreover, is consistent with the NMR data. These results illustrate the use of MD simulation at atomic resolution to model structural transitions and to interpret experimental data.     

Solution structures of alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.     

Saturation mutagenesis of a +1 programmed frameshift-inducing mRNA sequence derived from a yeast retrotransposon

Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the frequency up to 10,000-fold. These sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change in frames occurs along with additional sequences that stimulate the efficiency of frameshifting. One such stimulatory site derived from the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae (the Ty3 stimulator) comprises a 14 nucleotide sequence with partial complementarity to a Helix 18 of the 18S rRNA, a component of the ribosome's accuracy center. A model for the function of the Ty3 stimulator predicts that it base pairs with Helix 18, reducing the efficiency with which the ribosome rejects erroneous out of frame decoding. We have tested this model by making a saturating set of single-base mutations of the Ty3 stimulator. The phenotypes of these mutations are inconsistent with the Helix 18 base-pairing model. We discuss the phenotypes of these mutations in light of structural data on the path of the mRNA on the ribosome, suggesting that the true target of the Ty3 stimulator may be rRNA and ribosomal protein elements of the ribosomal entry tunnel, as well as unknown constituents of the solvent face of the 40S subunit.     

The water effect on the kinetic of the bovine liver catalase

Catalase is an enzyme that occurs in almost all aerobic organisms. Its main metabolic function is to prevent oxidative damage to tissues induced by hydrogen peroxide which is a strong oxidizing agent. Catalase is very effective in performing this task, since it has the highest turnover rate among all the enzymes. The properties of catalase have been investigated extensively for many years; however, the role of the solvent molecules in the catalytic reaction of this enzyme has not yet been investigated. Therefore, the objective of this work was to investigate the contribution of the solvent molecules on the catalytic reaction of bovine liver catalase with its substrate H2O2 by the osmotic stress method. As a probe for protein structural changes in solution, the differential number of water molecules released during the transition from free to bound form of the enzyme was measured. These assays were correlated with protein structural data provided by the SAXS technique and crystallographic structures of free and CN(-) bonded enzymes. The results showed that the difference in surface accessible area of the crystal structures does not reflect the variation that is observed in solution. Moreover, catalase is not influenced by the solvent during the catalytic reaction, which represents a lower energy barrier to be crossed in the overall energetics of the reaction, a fact that contributes to the high turnover rate of catalase.     

Role of subunit NuoL for proton translocation by respiratory complex I

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with a translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the electron transfer reaction and a membrane arm involved in proton translocation. The recently published X-ray structures of the complex revealed the presence of a unique 110 ? "horizontal" helix aligning the membrane arm. On the basis of this finding, it was proposed that the energy released by the redox reaction is transmitted to the membrane arm via a conformational change in the horizontal helix. The helix corresponds to the C-terminal part of the most distal subunit NuoL. To investigate its role in proton translocation, we characterized the electron transfer and proton translocation activity of complex I variants lacking either NuoL or parts of the C-terminal domain. Our data suggest that the H+/2e- stoichiometry of the ΔNuoL variant is 2, indicating a different stoichiometry for proton translocation as proposed from structural data. In addition, the same H+/e- stoichiometry is obtained with the variant lacking the C-terminal transmembraneous helix of NuoL, indicating its role in energy transmission.     

Ty1 in vitro integration: effects of mutations in cis and in trans.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.     

Ty1 transposition induced by carcinogens in Saccharomyces cerevisiae yeast depends on mitochondrial function

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).     

In vitro integration of retrotransposon Ty1: a direct physical assay.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.     

Electrostatic properties of bovine beta-lactoglobulin

Bovine beta-Lactoglobulin (BLG) has been studied for many decades, but only recently structural data have been obtained, making it possible to simulate its molecular properties. In the present study, electrostatic properties of BLG are investigated theoretically using Poisson-Boltzmann calculations and experimentally following pH titration via NMR. Electrostatic properties are determined for several structural models, including an ensemble of NMR structures obtained at low pH. The changes in electrostatic forces upon changes in ionic strength, solvent dielectric constant, and pH are calculated and compared with experiments. pK(a)s are computed for all titratable sites and compared with NMR titration data. The analysis of theoretical and experimental results suggests that (1) there may be more than one binding sites for negatively charged ligands; (2) at low pH the core of the molecule is more compact than observed in the structures obtained via restrained molecular dynamics from NMR data, but loop and terminal regions must be disordered.     

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant.

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.     

Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.     

A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 A, 1.85 A, 2.05 A and 2.2 A, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of single-site mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.     

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4–12 and 7–15, and its disulfide isomer, with disulfides positions 4–15 and 7–12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by ¹H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.     

Reliable structural interpretation of small-angle scattering data from bio-molecules in solution - the importance of quality control and a standard reporting framework

Small-angle scattering is becoming an increasingly popular tool for the study of bio-molecular structures in solution. The large number of publications with 3D-structural models generated from small-angle solution scattering data has led to a growing consensus for the need to establish a standard reporting framework for their publication. The International Union of Crystallography recently established a set of guidelines for the necessary information required for the publication of such structural models. Here we describe the rationale for these guidelines and the importance of standardising the way in which small-angle scattering data from bio-molecules and associated structural interpretations are reported.     

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum

Psalmopeotoxin I (PcFK1) is a 33-amino-acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra-erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function. We investigated the structural properties of PcFK1 to help understand the unique mechanism of action of this peptide and to enhance its utility as a lead compound for rational development of new antimalarial drugs. In this paper, we have determined the three-dimensional solution structure by (1)H two-dimensional NMR means of recombinant PcFK1, which is shown to belong to the ICK structural superfamily with structural determinants common to several neurotoxins acting as ion channels effectors.     

Mechanism of phosphoryl transfer catalyzed by shikimate kinase from Mycobacterium tuberculosis

The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate.