Biomedical subjects

A Dautant

Publications and source records attributed to A Dautant.

At least 19 recordsLinked to original sources

PubMed2001

Structure of mouse L-chain ferritin at 1.6 A resolution.

Cubic F432 crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique with ammonium sulfate and cadmium sulfate as precipitants. The structure was refined to 2.1 and 1.6 A resolution from data obtained at room temperature and under cryogenic conditions, respectively. The structure of an eight-amino-acid loop insertion in the mouse sequence is found to be highly disordered both at room temperature and at low temperature.

Animals↗

PubMed2000

Release of the cyano moiety in the crystal structure of N-cyanomethyl-N-(2-methoxyethyl)-daunomycin complexed with d(CGATCG).

Doxorubicin is among the most widely used anthracycline in cancer chemotherapy. In an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy, several analogues were synthesized. The cyanomorpholinyl derivative is the most cytotoxic. They differ greatly from their parent compound in their biological and pharmacological properties, inducing cross-links in drug DNA complexes. The present study concerns N-cyanomethyl-N-(2-methoxyethyl)-daunomycin (CMDa), a synthetic analogue of cyanomorpholino-daunomycin. Compared to doxorubicin, CMDa displays a cytotoxic activity on L1210 leukemia cells at higher concentration but is effective on doxorubicin resistant cells. The results of fluorescence quenching experiments as well as the melting temperature (DeltaTm = 7.5 degrees C) studies are consistent with a drug molecule which intercalates between the DNA base pairs and stabilizes the DNA double helix. The crystal structure of CMDa complexed to the hexanucleotide d(CGATCG) has been determined at 1.5 A resolution. The complex crystallizes in the space group P41212 and is similar to other anthracycline-hexanucleotide complexes. In the crystal state, the observed densities indicate the formation of N-hydroxymethyl-N-(2-methoxyethyl)-daunomycin (HMDa) with the release of the cyano moiety without DNA alkylation. The formation of this degradation compound is discussed in relation with other drug modifications when binding to DNA. Comparison with two other drug-DNA crystal structures suggests a correlation between a slight change in DNA conformation and the nature of the amino sugar substituents at the N3' position located in the minor groove.

Animals↗

PubMed2000

Crystallization and preliminary X-ray diffraction data of mouse L-chain apoferritin crystals.

Crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique using ammonium sulfate as precipitant. Two crystal forms were observed in the same drop. The crystals belong to either the P2 monoclinic or to the P42(1)2 tetragonal space group. The monoclinic crystals diffracted to beyond 2.4 A resolution but were systematically twinned, while the tetragonal crystals diffracted to beyond 2.9 A. These crystallization conditions in the absence of metal salts should facilitate the study of the interaction between L-chain ferritins and heavy metals, particularly the iron core.

Animals↗

PubMed1999

Expression, purification, crystallization and preliminary X-ray diffraction results from Campylobacter jejuni ferritin.

The prokaryotic ferritin gene of Campylobacter jejuni was overexpressed in Escherichia coli under control of the bacteriophage T7 promoter and the protein (Cj-FTN) purified. Preliminary crystallization experiments have been performed using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction studies show the crystals belong to the I432 space group (a = 151.52 A). Structure solution by molecular replacement is in progress while crystal quality improvement is carried out.

Campylobacter jejuni↗

PubMed1998

Evidence of new cadmium binding sites in recombinant horse L-chain ferritin by anomalous Fourier difference map calculation.

We refined the structure of the tetragonal form of recombinant horse L-chain apoferritin to 2.0 A and we compared it with that of the cubic form previously refined to the same resolution. The major differences between the two structures concern the cadmium ions bound to the residues E130 at the threefold axes of the molecule. Taking advantage of the significant anomalous signal (f" = 3.6 e-) of cadmium at 1.375 A, the wavelength used here, we performed anomalous Fourier difference maps with the refined model phases. These maps reveal the positions of anomalous scatterers at different locations in the structure. Among these, some are found near residues that were known previously to bind metal ions, C48, E57, C126, D127, E130, and H132. But new cadmium binding sites are evidenced near residues E53, E56, E57, E60, and H114, which were suggested to be involved in the iron loading process. The quality of the anomalous Fourier difference map increases significantly with noncrystallographic symmetry map averaging. Such maps reveal density peaks that fit the positions of Met and Cys sulfur atoms, which are weak anomalous scatterers (f" = 0.44 e-).

Animals↗

PubMed1998

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human uroporphyrinogen decarboxylase.

A recombinant human uroporphyrinogen decarboxylase (E.C. 4.1.1.37, UROD) has been expressed in Escherichia coli and purified to homogeneity. Crystals grew by the hanging-drop vapor-diffusion technique from a starting solution containing 1.5 mg ml-1 protein. The crystals belong to the trigonal space group P3121 or its enantiomer P3221 and diffract to 3 A resolution. The unit-cell parameters are a = b = 103.4, c = 75.7 A and gamma = 120 degrees. The asymmetric unit contains one molecule. Preliminary structural predictions suggest for the protein a TIM-barrel type tertiary structure.

Crystallization↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1997

Comparison of the structures of the cubic and tetragonal forms of horse-spleen apoferritin.

Horse-spleen apoferritin is known to crystallize in three different space groups, cubic F432, tetragonal P42(1)2 and orthorhombic P2(1)2(1)2. A structure comparison of the cubic and tetragonal forms is presented here. Both crystal forms were obtained by the vapor-diffusion technique and data were collected at 2.26 A (cubic crystal) and 2.60 A (tetragonal crystal) resolution. Two main differences were observed between these crystal structures: (i) whereas intermolecular contacts only involve salt-bridge type interactions via cadmium ions in the cubic structure, two types of interactions are observed in the tetragonal crystal (cadmium-ion-mediated salt bridges and hydrogen-bonding interactions) and (ii) cadmium ions bound in the threefold axes of ferritin molecules exhibit lower site-occupation factors in the tetragonal structure than in the cubic one.

Journal Article↗

PubMed1996

Parallel and antiparallel (G.GC)2 triple helix fragments in a crystal structure.

Nucleic acid triplexes are formed by sequence-specific interactions between single-stranded polynucleotides and the double helix. These triplexes are implicated in genetic recombination in vivo and have application to areas that include genome analysis and antigene therapy. Despite the importance of the triple helix, only limited high-resolution structural information is available. The x-ray crystal structure of the oligonucleotide d(GGCCAATTGG) is described; it was designed to contain the d(G middle dotGC)2 fragment and thus provide the basic repeat unit of a DNA triple helix. Parameters derived from this crystal structure have made it possible to construct models of both parallel and antiparallel triple helices.

Base Composition↗

PubMed1996

Formation of (C.G)*G triplets in a B-DNA duplex with overhanging bases.

Crystallization of a DNA double helix with overhanging bases at the 5'-ends of both strands, results in the formation of two crystallographically independent (C.G)*G triplets. In a previous report [Van Meervelt, Vlieghe, Dautant, Gallois, Précigoux & Kennard (1995). Nature (London), 374, 742-744] the unique molecular packing of the duplex and the Hoogsteen hydrogen-bond pattern and parallel backbone orientation of the guanine-containing strands in the triplets was described. The fine structural details and hydration of the d(GCGAATTCG) crystal structure refined to 2.05 A (R = 0.168, 86 water molecules, two Mg(2+) cations) are now presented. Helical parameters, stacking effects, the geometry at the duplex-triplex junction, and the hydration of the minor groove are discussed and compared with related theoretical and crystal structures.

Journal Article↗

PubMed1996

Preliminary X-ray diffraction studies of the tetragonal form of native horse-spleen apoferritin.

Horse-spleen ferritin is known to crystallize in three different space groups, cubic F432, orthorhombic P2(1)2(1)2 and tetragonal P42(1)2, but only the cubic form has been fully investigated. Crystals of the tetragonal form of apoferritin have been obtained, by the vapour-diffusion technique, which diffract beyond 3.0 A. The unit-cell dimensions are a = b = 146.63, c = 152.94 A. The orientation of the non-crystallographic symmetry axes of the apoferritin molecule (24 subunits of 174 amino acids each, arranged in a 432 point symmetry rhombododecahedron) has been determined by a self-rotation Patterson function. The asymmetric unit is made of six subunits and was positioned by molecular replacement.

Journal Article↗

PubMed1996

Crystallization and preliminary X-ray analysis of an orthorhombic form of horse-spleen apoferritin.

Horse-spleen apofemtin crystallizes in two different space groups: cubic F432 and tetragonal P42(1)2 while its iron-containing analogue is known to present a cubic and an orthorhombic form. Up to now, only the structure of the cubic form has been fully investigated by X-ray diffraction, although some information concerning the molecular packing of the two other forms was deduced from analysis of X-ray photographs. While growing cubic crystals of horse-spleen apoferritin with Pt-mesoporphyrin IX, we obtained one crystal, with a diffraction limit of 2.4 A, belonging to the orthorhombic P2(1)2(1)2 space group, with unit-cell dimensions a = 181.6, b = 128.9, c = 128.9 A. The orientation of the non-crystallographic axes of the molecule was determined by self-rotation Patterson function and the structure was determined by the molecular-replacement method. The asymmetric unit consists of half an apoferritin molecule. Refinement of the structure is in progress, some preliminary results of the molecular packing are given.

Journal Article↗

PubMed1996

Structural investigation of the complexation properties between horse spleen apoferritin and metalloporphyrins.

Crystallographic studies of L-chain horse spleen apoferritin (HSF) co-crystallized with Pt-hematoporphyrin IX and Snprotoporphyrin IX have brought significant new insights into structure-function relationships in ferritins. Interactions of HSF with porphyrins are discussed. Structural results show that the nestling properties into HSF are dependent on the porphyrin moiety. (Only protoporphyrin IX significantly interacts with the protein, whereas hematoporphyrin IX does not.) These studies additionally point out the L-chain HSF ability to demetalate metalloporphyrins, a result which is of importance in looking at the iron storage properties of ferritins. In both compound investigated (whether the porphyrin reaches the binding site or not), the complexation appears to be concomitant with the extraction of the metal from the porphyrin. To analyze further the previous results, a three-dimensional alignment of ferritin sequences based on available, crystallographic coordinates, including the present structures, is given. It confirms a high degree of homology between these members of the ferritin family and thus allows us to emphasize observed structural differences: 1) unlike L-chain HSF, H-chain human ferritin presents no preformed binding site; and 2) despite the absence of axial ligands, and due to the demetalation, L-chain HSF is able to host protoporphyrin at a similar location to that naturally found in bacterioferritin.

Amino Acid Sequence↗

PubMed1995

A trigonal form of the idarubicin:d(CGATCG) complex; crystal and molecular structure at 2.0 A resolution.

The X-ray crystal structure of the complex between the anthracycline idarubicin and d(CGATCG) has been solved by molecular replacement and refined to a resolution of 2.0 A. The final R-factor is 0.19 for 3768 reflections with Fo > or = 2 sigma (Fo). The complex crystallizes in the trigonal space group P31 with unit cell parameters a = b = 52.996(4), c = 33.065(2) A, alpha = beta = 90 degree, gamma = 120 degree. The asymmetric unit consists of two duplexes, each one being complexed with two idarubicin drugs intercalated at the CpG steps, one spermine and 160 water molecules. The molecular packing underlines major groove-major groove interactions between neighbouring helices, and an unusually low value of the occupied fraction of the unit cell due to a large solvent channel of approximately 30 A diameter. This is the first trigonal crystal form of a DNA-anthracycline complex. The structure is compared with the previously reported structure of the same complex crystallizing in a tetragonal form. The geometry of both the double helices and the intercalation site are conserved as are the intramolecular interactions despite the different crystal forms.

Base Sequence↗

PubMed1995

High-resolution structure of a DNA helix forming (C.G)*G base triplets.

Triple helices result from interaction between single- and double-stranded nucleic acids. Their formation is a possible mechanism for recombination of homologous gene sequences in nature and provides, inter alia, a basis for artificial control of gene activity. Triple-helix motifs have been extensively studied by a variety of techniques, but few high-resolution structural data are available. The only triplet structures characterized so far by X-ray diffraction were in protein-DNA complexes studied at about 3 A resolution. We report here the X-ray analysis of a DNA nonamer, d(GCGAATTCG), to a resolution of 2.05 A, in which the extended crystal structure contains (C.G)*G triplets as a fragment of triple helix. The guanosine-containing chains are in a parallel orientation. This arrangement is a necessary feature of models for homologous recombination which results ultimately in replacement of one length of DNA by another of similar sequence. The present-structure agrees with many published predictions of triplex organization, and provides an accurate representation of an element that allows sequence-specific association between single- and double-stranded nucleic acids.

Base Sequence↗

PubMed1994

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.

Journal Article↗

PubMed1994

Onset of the fully extended conformation in (alpha Me)Leu derivatives and short peptides.

The X-ray diffraction crystal structures of the (alpha Me)Leu derivative mClAc-D-(alpha Me) Leu-OH and the terminally protected tripeptide Z-D-(alpha Me) Leu-(L-Ala)2-OMe show the onset of the fully extended (C5) conformation for the (alpha Me) Leu residue in both independent molecules in the asymmetric unit of the former compound and in two out of the four independent molecules in the asymmetric unit of the latter compound. In addition, conformational analysis in CDCl3 solution (using FT-infra-red absorption and 1H nuclear magnetic resonance) revealed the occurrence of a significant population of fully extended conformers throughout the entire sequence of the (alpha Me) Leu homochiral homopeptides pBrBz-[D-(alpha Me) Leu]n-OtBu (from monomer to tetramer). Taken together, these results represent a clear indication that this peptide secondary structure, uncommon for protein amino acids and other C alpha-methylated chiral residues, is not a rare observation in (alpha Me) Leu derivatives and short peptides.

Amino Acid Sequence↗

PubMed1993

Modelling, synthesis and biological activity of a BLV proteinase, made of (only) 116 amino acids.

Bovine leukaemia virus (BLV) is the aetiological agent of Leukosis enzootica bovis [Viral Oncology (1980), G. Klein (Ed.) Raven Press, New York, pp. 231-238], a widely spread disease in cattle. BLV is reported as the animal model of human T-cell leukaemia virus (HLTV) which is the causative agent of adult T-cell leukaemia and tropical spastic paraparesis. Like the viruses themselves, the two retroviral proteinases (PR) are very closely related [Virology 142 (1985) 357-377]. BLV and HTLV-I PR are reported as putative proteins made of 126 [J. Virol. 57 (1986) 826-832] and 125 [FEBS Lett. 293 (1991) 106-110] amino acids, respectively (long sequences), belonging to the aspartyl proteinase family [Nature 329 (1987) 351-354], with the aid of molecular modelling, we show that BLV and HTLV-I proteinases made of only 116 and 115 amino acids, respectively (short sequences), display three-dimensional structures similar to that observed for other retroviral aspartyl proteinases. The models are based on three-dimensional structures of Rous sarcoma virus (RSV PR) and the human immunodeficiency virus (HIV-1 PR). We used solid phase peptide synthesis to produce the putative proteolytic enzyme of BLV (116 amino acids). In this study, we show that the folded synthetic protease accurately hydrolyzes a decapeptide corresponding to the sequence of the Matrice-Capside (MA/CA) cleavage site of the gag polyprotein. In addition, the proteolytic activity is inhibited by a statine ((4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid) containing an analogous sequence.

Amino Acid Sequence↗