Denise Sy

Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway

BACKGROUND In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols. Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain. This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme iron dependent enzyme that is active as a homotetramer in bacteria and as a homodimer in plants. Moreover, in humans, a HPPD deficiency is found to be related to tyrosinemia, a rare hereditary disorder of tyrosine catabolism. RESULTS We report here the crystal structure of Pseudomonas fluorescens HPPD refined to 2.4 A resolution (Rfree 27.6%; R factor 21.9%). The general topology of the protein comprises two barrel-shaped domains and is similar to the structures of Pseudomonas 2,3-dihydroxybiphenyl dioxygenase (DHBD) and Pseudomonas putida catechol 2,3-dioxygenase (MPC). Each structural domain contains two repeated betaalpha betabeta betaalpha modules. There is one non-heme iron atom per monomer liganded to the sidechains of His161, His240, Glu322 and one acetate molecule. CONCLUSIONS The analysis of the HPPD structure and its superposition with the structures of DHBD and MPC highlight some important differences in the active sites of these enzymes. These comparisons also suggest that the pyruvate part of the HPPD substrate (4-hydroxyphenylpyruvate) and the O2 molecule would occupy the three free coordination sites of the catalytic iron atom. This substrate-enzyme model will aid the design of new inhibitors of the homogentisate biosynthesis reaction.

Radioprotection of DNA by spermine: a molecular modelling approach

PURPOSE To observe and explain the sequence-dependence of DNA radioprotection by spermine. MATERIALS AND METHODS Sequencing gel electrophoresis was used to analyse the probability of frank strand break (FSB) induction at each nucleotide site. Molecular modelling of complexes of DNA with spermine molecules and of a curved electrically null DNA has been performed. RESULTS The effect of spermine on radiation-induced strand breakage varied significantly along the studied fragment. At low spermine concentration, some sequences were protected while others were unprotected. Molecular modelling calculations show that the most electro-negative sites are located in the minor or in the major groove of DNA. The positively charged spermine (Z=+4) should preferentially bind to such sites. When bound in the minor groove, spermine triggers a reduction of the accessibility of radiolytic attack sites to OH* radicals. This is due to induced structural modifications and to the masking of attack sites. In the case of major groove binding, no reduction of accessibility occurs. This type of binding can explain the lack of protection of sequences with electro-negative sites in the major groove. At high spermine concentration, the fragment is strongly protected. A nucleosome-like pattern of breakage with periodically distributed regions of protection was observed. Molecular modelling calculations show that the accessibility of the attack sites in a curved electrically null DNA is also periodically reduced. CONCLUSIONS Molecular modelling of DNA-spermine complexes that takes into account the electrostatic properties of DNA, allows an explanation of the experimentally observed effects of spermine on DNA radiosensitivity.

Androctonin, a novel antimicrobial peptide from scorpion Androctonus australis: solution structure and molecular dynamics simulations in the presence of a lipid monolayer.

Androctonin is a highly cationic antimicrobial peptide from scorpion exhibiting a broad spectrum of activities against bacteria and fungi. It contains 25 amino acids including four cysteine residues forming two disulfide bridges. We report here on the determination of its solution structure by conventional two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling using distance geometry and molecular dynamics methods. The structure of androctonin involves a well-defined highly twisted anti-parallel beta-sheet with strands connected by a more variable positively charged turn. A comparison with the structure of tachyplesin I (horseshoe crab) reveals that the amphiphilic character of the protein surface of this homologous peptide is not observed in androctonin. We have undertaken a 200-ps molecular dynamics simulation study on a system including one androctonin molecule and a monolayer of DMPG (1,2-dimyristoylphosphatidylglycerol) lipids. On the basis of this simulation, the first steps of the membrane permeabilization process are discussed.

RADACK, a stochastic simulation of hydroxyl radical attack to DNA.

Abstract RADACK was conceived to simulate the radiation-induced attack to different DNA forms and complexes. It allows to separately calculate the probability of attack to each reactive atom of the sugar and of the base and takes into account the sequence-dependent structure of DNA as known from crystallographic or NMR studies or resulting from molecular modelling. The calculations are aimed to assess sequence-, structure- and ligand-dependent modulation of damages of sugar and bases, leading to single strand breaks (frank strand breaks, FSB) and alkali-labile base modifications (alkali-revealed breaks, ARB), respectively. The modelling procedure and the results of simulations for some representative structures (B, Z and quadruplex forms) are here described and discussed. The calculated relative probabilities of OH. radical attack to all reaction sites are compared to experimental FSB and ARB values. By a fitting procedure, the relative efficiencies of conversion of the C4′ and C5′-centred radicals into FSB, ϵ (C4′): ϵ (C5′), and the relative efficiencies of base radicals—to—ARB conversion, ϵ (T): ϵ(A): ϵ(C): ϵ(G), are then deduced for each DNA form. The ability of the model to account for the distribution of damages in DNA-ligand complexes is proven by its successful application to two DNA-protein systems: the lac repressor-lac operator complex and the nucleosome core.

Radiation Disrupts Protein–DNA Complexes through Damage to the Protein. The lac Repressor–Operator System

Abstract Eon, S., Culard, F., Sy, D., Charlier, M. and Spotheim-Maurizot, M. Radiation Disrupts Protein–DNA Complexes through Damage to the Protein. The lac Repressor–Operator System. Radiat. Res. 156, 110–117 (2001). Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein–DNA recognition, the entire regulation process may be perturbed. We have studied the effect of γ rays on a model regulatory system, the E. coli lactose repressor–operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, but not by the addition of nonirradiated DNA. Thus radiation disrupts the DNA–protein complex by affecting the binding ability of the protein. This interpretation is supported by the dramatic loss of binding ability of a free irradiated repressor toward nonirradiated DNA. Interestingly, the dose necessary for the disruption of the irradiated complex is higher than that for inducing the complete loss of the binding ability of the free irradiated repressor. This may be due to the protection of key amino acids by the bound DNA. As seen from calculations of the accessibility of amino acids to radiolytic OH·, the protection is due to both masking and conformational effects.

In Vitro Studies with Methylproamine A Potent New Radioprotector

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is ∼100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)2 confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.

Protein Stability and Plasticity of the Hydrophobic Cavity in Wheat ns-LTP

Abstract Plant ns-LTPs display an original structure with four helices and a flexible C-terminus, maintained together by four disulphide bridges and delineating an elongated central hydrophobic cavity. In order to relate these structural features to the protein stability and plasticity, combined molecular mechanics and simulated annealing calculations were undertaken on a wheat ns-LTP “mutant” with Cys-Ala replacement and with the application of core inter-residue restraints up to 2 Å, reducing the cross-section size of the hydrophobic cavity. Analysis of the energy-minimized structures shows that removal of the disulphide bridges results in structures with a lower total energy and a smaller cavity volume. A 1-ns MD simulation at 300K in water, underlines that, despite the absence of a well-packed hydrophobic core, the native structure is extremely stable at room temperature and the cavity is not hydrated. This confirms that the disulphide bridges are essential for the existence of the cavity, whereas its plasticity depends both on the hydrophobic chain lining the cavity and on the C-terminal flexibility. A high temperature (500K) MD simulation confirms the stability of the secondary structure elements and the flexibility of the loops and of the C-terminal segment. Two important structural transitions during this simulation are discussed and possible routes for the insertion and release of hydrophobic ligands are suggested.

Radiolysis of nucleosome core DNA: a modelling approach

PURPOSE To calculate the expected pattern of frank strand breaks (FSB) induced in the DNA of a nucleosome core particle (NCP) by the attack of radiolytic OH* radicals and to compare this with the experimental pattern of FSB induced by the in vitro irradiation of chicken NCP. MATERIALS AND METHODS The structure of NCP was obtained from the PDB databank. Using molecular modelling, the structure of a linear DNA fragment with the central 60 bp sequence of NCP-DNA was determined. The accessibility of the sugar attack sites to OH* radicals at each nucleotide of the linear fragment or of the NCP-DNA was calculated. The probability of reaction of OH* with each sugar reactive site was calculated using a Monte-Carlo method-based stochastic model. RESULTS The accessibility of attack sites in the NCP-DNA and the calculated probabilities of sugar attack are mainly modulated through masking by histones, and only slightly through bending. The periodicity of the calculated FSB probabilities is identical to that of the experimental FSB probabilities in chicken NCP (period 10.4 +/- 0.1 bp). CONCLUSIONS The good agreement between the calculated and experimental results recommends the presented modelling procedure as a tool in predicting the radiosensitivity of DNA in DNA-ligand complexes of known structure.Purpose : To calculate the expected pattern of frank strand breaks (FSB) induced in the DNA of a nucleosome core particle (NCP) by the attack of radiolytic OH •radicals and to compare this with the experimental pattern of FSB induced by the in vitro irradiation of chicken NCP. Materials and methods : The structure of NCP was obtained from the PDB databank. Using molecular modelling, the structure of a linear DNA fragment with the central 60 bp sequence of NCPDNA was determined. The accessibility of the sugar attack sites to OH •radicals at each nucleotide of the linear fragment or of the NCP-DNA was calculated. The probability of reaction of OH •with each sugar reactive site was calculated using a MonteCarlo method-based stochastic model. Results : The accessibility of attack sites in the NCP-DNA and the calculated probabilities of sugar attack are mainly modulated through masking by histones, and only slightly through bending. The periodicity of the calculated FSB probabilities is identical to that of the experimental FSB probabilities in chicken NCP (period 10.4 ±0.1bp). Conclusions : The good agreement between the calculated and experimental results recommends the presented modelling procedure as a tool in predicting the radiosensitivity of DNA in DNA-ligand complexes of known structure.

DNA radiolysis in DNA–protein complexes: a stochastic simulation of attack by hydroxyl radicals

Purpose: To propose an improved version of RADACK, a stochastic simulation of radiolytic attack on DNA, that takes into account the reactivity of each amino acid of a specifically bound protein with hydroxyl radicals. To apply it to the natural lactose operator–repressor complex taking advantage of recently reported structures. To compare the obtained probabilities of DNA strand break induction with those calculated with the previous versions and with an experimental pattern of strand break probabilities. Materials and methods: Models of complexes close to the natural ones, derived from crystallography‐ and NMR‐based structures recently available in the PDB databank, were used. The specific chemical reactivity of each amino acid was introduced in the new version of RADACK (the reactivity model). The probabilities of strand break induction by the irradiation of the complex were calculated with this new version as well as with previous ones. Results: The patterns of probabilities of strand break induction calculated with the improved version of RADACK were partially different from those obtained with previous versions. The patterns obtained for both, using putative models of natural complexes, were consistent with the experimental results, but some discrepancies were suggestive of slight differences between these structures and the real natural system. The crystallographic structure agreed best with the experimental results. Conclusions: A new version of RADACK was validated that took into account the reactivity of atoms in both DNA and protein. The putative modelled structures of a natural lactose operator–repressor complex were discussed.

E.S.R. Investigation of Spin Probes: Not Unique Ordering in Smectic Mesophases

Abstract E.S.R behaviour of various nitroxide probes dissolved in two mesomorphic compounds of the homologous series methoxy-benzylidene-alkyl-cinnamates is investigated. Probe alignment by the mesomorphic host molecules is not unique in smectic phases; intercalation of the probe between the smectic layers occurs, depending on the comparative strength of lateral and interlayer correlations.