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.

Initiation of DNA replication by DNA polymerases from primers forming a triple helix.

Despite extensive studies on oligonucleotide-forming triple helices, which were discovered in 1957, their possible relevance in the initiation of DNA replication remains unknown. Using sequences forming triple helices, we have developed a DNA polymerisation assay by using hairpin DNA templates with a 3' dideoxynucleotide end and an unpaired 5'-end extension to be replicated. The T7 DNA polymerase successfully elongated nucleotides to the expected size of the template from the primers forming triple helices composed of 9-14 deoxyguanosine-rich residues. The triple helix-forming primer required for this reaction has to be oriented parallel to the homologous sequence of the hairpin DNA template. Substitution of the deoxyguanosine residues by N7 deazadeoxyguanosines in the hairpin of the template prevented primer elongation, suggesting that the formation of a triple helix is a prerequisite for primer elongation. Furthermore, DNA sequencing could be achieved with the hairpin template through partial elongation of the third DNA strand forming primer. The T4 DNA polymerase and the Klenow fragment of DNA polymerase I provided similar DNA elongation to the T7 polymerase-thioredoxin complex. On the basis of published crystallographic data, we show that the third DNA strand primer fits within the catalytic centre of the T7 DNA polymerase, thus underlying this new property of several DNA polymerases which may be relevant to genome rearrangements and to the evolution of the genetic apparatus, namely the DNA structure and replication processes.

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.

Inhibition of activity of the protease from bovine leukemia virus.

In view of the close similarity between bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I) we investigated the possibility of developing specific inhibitors of the proteases of these retroviruses using the purified enzyme from BLV. We tested the ability of this protease to specifically cleave various short oligopeptide substrates containing cleavage sites of BLV and HTLV-I proteases, as well as a recombinant BLV Gag precursor. The best substrate, a synthetic decapeptide bearing the natural cleavage site between the matrix and the capsid proteins of BLV Gag precursor polyprotein, was used to develop an inhibition assay. We determined the relative inhibitory effect of synthetic Gag precursor-like peptides in which the cleavable site was replaced by a non-hydrolyzable moiety. The encouraging inhibitory effect of these compounds indicates that potent non-peptidic inhibitors for retroviral proteases are not unattainable.

Inhibition of prostaglandin synthetase by di- and triphenylethylene derivatives: a structure-activity study.

The syntheses of new di- and triphenylethylene derivatives are described along with their X-ray analysis and NMR study, which have helped to establish their conformation. Screening of over 50 derivatives for inhibition of prostaglandin synthetase (PGS) activity in bovine seminal vesicle microsomes has revealed that many of the triphenylethylene derivatives are potent inhibitors of PGS. Several even show marked activity at the extremely low concentration (IC50) of about 4 X 10(-8) M, which is two orders of magnitude lower than the active concentration of the majority of known nonsteroidal antiinflammatory agents (IC50 approximately equal to 10(-6) M). Unlike the latter, these compounds are not carboxylic acids. Furthermore, in contrast to biphenyl, diphenylmethane, or unsymmetrical, alpha, alpha'-diphenylethylene PGS inhibitors, the presence of a beta-phenyl ring was an essential requirement for high potency. The best inhibitors possessed a cyanide group (acids, amides, and amines were poor inhibitors), methoxy in preference to hydroxy groups on the alpha-phenyl rings, and a halogen (F or Cl) in a para position on the beta-phenyl ring. These data provide additional insight into the nature of the PGS binding site.

Reverse relationship between alpha-carbon chirality and helix handedness in (alpha Me)Phe peptides.

The crystal-state preferred conformations of two tripeptides, one tetrapeptide, and one pentapeptide, each containing a single residue of the chiral, C alpha, alpha-disubstituted glycine C alpha-methyl, C alpha-benzylglycine [(alpha Me)Phe], have been determined by X-ray diffraction. The tripeptides are Z-L-(alpha Me)Phe-(Aib)2-OH dihydrate and Z-Aib-D-(alpha Me)Phe-Aib-OtBu, the tetrapeptide is Z-(Aib)2-D-(alpha Me)Phe-Aib-OtBu, and the pentapeptide is pBrBz-(Aib)2-DL-(alpha Me)Phe-(Aib)2-OtBu. While the two tripeptides are folded in a beta-bend conformation, two such conformations are consecutively formed by the tetrapeptide. The pentapeptide adopts a regular 3(10)-helix promoted by three consecutive beta-bends. This study confirms the strong propensity of short peptides containing C alpha-methylated alpha-aminoacids to fold into beta-bends and 3(10)-helical structures. Since Aib is achiral, the handedness of the observed bends and helices is dictated by the presence of the (alpha Me)Phe residue. In general, we have found that the relationship between (alpha Me)Phe chirality and helix handedness is opposite to that exhibited by protein aminoacids. A comparison with the preferred conformation of other extensively investigated C alpha-methylated aminoacids is made.

[Cardiovascular effects of peptide inhibitors of the renin-substrate reaction in rats with renovascular hypertension. Goldblatt: 2 kidneys--1 clip]. / Effets cardio-vasculaires d'inhibiteurs peptidiques de la réaction rénine-substrat chez le rat atteint d'hypertension réno-vasculaire. Goldblatt: 2 reins--1 clip.

The antihypertensive effects of 2 different peptidic substrate analogs: AG 84-10 AG 85-12 were investigated in renovascular hypertensive (Goldblatt, 2 kidneys--1 clip) Sprague-Dawley male rats. AG 84-10 (Ac-Pro-Phe-His-Leu-Val-Tyr) is similar to Angiotensinogen 6-13 and AG 85-12 (Ac-Ile-His-Pro-Phe-His-Leu) mimics the C-terminal portion of Angiotensin I. 6 weeks after clipping, hemodynamic profiles of these molecules [Heart rate (HR), mean arterial pressure (MAP), filling parameters, peripheral vascular resistances (PR) and cardiac output (CO)] during 90 minutes, were determined in the anesthetized animals. CO was measured using a thermodilution technique. Parallel radio-immunologic dosages of plasma renin activity were performed. Measurements and calculation of previously defined hemodynamic variables, every 10 minutes, demonstrated that: AG 84-10 exerted an early but transient decrease of MAP and PR, an increase of CO without modification of other hemodynamic parameters. AG 85-12 induced a late and durable decrease of MAP and PR with a significant decrease of heart rate, but without modification of CO and other hemodynamic variables. Example: PR mmHg/ml/mn/kg (mean +/- SD): *p less than 0.05 ** p less than 0.01. (Table: see text). The different levels of plasma renin activity were in accordance with hemodynamic data. So, the 2 peptidic substrate analogs elicited antihypertensive effects with a more efficient action of AG 85-12.

Conformational preferences and the role of the statine residue in the crystal state.

The present paper is the result of an analysis of the available crystal structure data related to the statine amino acid so as to obtain information about bond lengths, bond angles, and preferential conformations. The number of configurations actually observed is small; nevertheless, some characteristic conformations should be pointed out for statine-containing peptides. The presence of two additional carbon atoms in the statine main chain enhances the peptide conformational degree of freedom and the statine-containing peptides are observed in a variety of different conformations including some usual secondary structure-types as beta-turns and beta-strands.

The renin-angiotensin system: an example of the study of linear peptides by x-ray crystallography.

In order to get information on the bioactive conformations of the endogenic renin substrate, a few peptide segments of angiotensinogen, along with a pepstatin analogue, were studied in the solid state by x-ray crystallography. These results are compared with the conformations of acidic proteinase inhibitors observed at the level of the active site. Such a comparison allows us to point out some analogies and differences between the observed conformation for the peptide alone and the conformations on the active sites. The analysis of the results should be a good starting point for making hypotheses on the renin substrate bioactive conformation(s).

X-ray analysis of renin substrates. Phenyloxyacetyl-leucyl-valyl-phenylalanine methyl ester: an analogue of angiotensinogen-(10-13) sequence.

The crystal structure of the title compound, an analogue of the angiotensinogen-(10-13) peptide in which the N-terminal leucine and the C-terminal tyrosine are respectively replaced by the phenyloxy-acetic group and by phenylalanine, has been determined by X-ray diffraction. The peptide crystallizes in the space group P2(1)2(1)2(1) with a = 4.866(1), b = 22.311(3), c = 27.213(4) A and Z = 4. The crystal structure was solved by direct methods and refined to an R value of 0.056. The molecules adopt a pleated sheet conformation with the hydrophobic residues alternatively situated on the right and left of the main chain. In the crystallographic "a" direction, the molecules are linked by hydrogen bonds and form parallel pleated sheet-type structures.

Influence of thermal motion on 1H chemical shifts in proteins: the case of bovine pancreatic trypsin inhibitor.

The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C(alpha)-C(beta) bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared.

Crystal structure and conformational analysis of angiotensinogen fragments.

The tripeptide acetyl-L-prolyl-L-phenylalanyl-L-histidine crystallizes in the orthorhombic space group P2(1)2(1)2(1) with eight molecules in a unit cell of dimensions a = 9.028(2), b = 140.54(6) and c = 42.41(1)A. The structure has been solved by direct methods and refined to an R value of 0.056 for 2904 observed reflections. The molecule exists as a zwitterion with terminal (His)CO2- and (imidazole)H+ as charged groups. The two peptide molecules in the structure adopt a type I beta-turn with Pro and Phe as the corner residues. The main conformational difference between the two crystallographically independent molecules is seen to be in the histidine side-chain orientations. The molecules arrange themselves in sheets perpendicular to the c axis. All hydrophobic side chains lie on one side of the sheets thus generated, whereas the hydrophilic groups are located on the other side. An interesting feature of the crystal structure is the existence of a water layer between adjacent peptide sheets. The conformational study of the isolated Ac-His-Pro-Phe-His-MA using energy calculations gives a rather limited number of stable conformers. The most stable corresponds to a type I beta-turn stabilized through two hydrogen bonds, followed by a less stable type II beta-turn (delta E = 2.0 kcal) and a partly helical structure (delta E = 2.6 kcal).

[Homologous radioimmunoassay of a hypothalamic factor stimulating salmon pituitary gonodatropic function (sGnRH)]. / Dosage radio-immunologique homologue d'un facteur hypothalamique de stimulation de la fonction gonadotrope hypophysaire de saumon s-Gn-RH.

A radioimmunoassay for Salmon Gn-RH p-gly-His-Trp-Ser-Tyr-Gly-Trp- Leu-Pro-Gly) (NH2) has been developed with a sensitivity of 7 pg/assay tube. The system allows the specific detection of an immunological GnRH related substance in the brain and pituitary of three teleost species but not in an elasmobranch the Dogfish. these results are discussed and some Gn-RH contents of the organs are proposed.

Monoclinic uncomplexed double-stranded, antiparallel, left-handed beta 5.6-helix (increases decreases beta 5.6) structure of gramicidin A: alternate patterns of helical association and deformation.

A comparison of the monoclinic and orthorhombic crystal structures of the uncomplexed double-stranded, antiparallel, left-handed beta-helix (5.6 amino acid residues per turn) (increases decreases beta 5.6) conformers of gramicidin A reveals marked differences in the tryptophan side-chain orientations and the degree of helical uniformity of the dimer and in the manner in which these helical dimers associate with one another in the crystal. The helix of the orthorhombic dimer exhibits a regular pattern of bulges and constrictions that appears to be induced by crystal packing forces affecting tryptophan side chains that are aligned parallel to the helix axis. The monoclinic dimer is more uniform than the orthorhombic dimer as a consequence of pi stacking interactions between dimers in which orientation of tryptophan side chains is normal to the helix axis to relieve the lateral crystal packing forces that may locally twist and deform the helix. It may be inferred from these observations that lipid interactions may be expected to destabilize the increases decreases beta 5.6 helix when it is inserted into a membrane bilayer.

Orthorhombic crystal structure of the A-DNA octamer d(GTACGTAC). Comparison with the tetragonal structure.

The X-ray crystal structure of the double-helical A-DNA octanucleotide d(GTACGTAC) has been solved by molecular replacement and refined to a resolution of 0.219 nm. The final R-factor is equal to 16.1% for 1516 observed reflections with F > 4 sigma(F). The sequence crystallizes as an A-DNA-type double helix in the orthorhombic space group P2(1)2(1)2, with one duplex molecule solvated by 66 water molecules in the asymmetric unit. Cell parameters are a = 3.860 nm, b = 5.082 nm, c = 2.174 nm. It is the first time that such a crystal form has been observed. This orthorhombic structure has been compared with the tetragonal structure of the same oligonucleotide. It adopts a bent structure with an unusual packing between symmetry-related molecules.

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.

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.

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.

Immunoreactive gonadotropin-releasing hormone-like material in the brain and the pituitary gland during the periovulatory period in the brown trout (Salmo trutta L.): relationships with the plasma and pituitary gonadotropin.

In fish there are few data on the gonadotropin-releasing hormone (Gn-RH) neurosecretory activity, which could explain long- and short-term variations of the gonadotropin secretion. There is no biological species specificity between mammal and fish Gn-RH; although there is a structural difference, they are, on the contrary, characterized by a high immunological specificity which does not allow measurement of fish Gn-RH using radioimmunoassay for LH-RH. We have synthesized salmon Gn-RH according to the formula recently proposed by Sherwood (N. Sherwood, L. Eiden, M. Brownstein, J. Spies, J. Rivier, and W. Vale, 1983. Proc. Natl. Acad. Sci. USA 80, 2794-2798). Its activity has been tested by its ability to stimulate the gonadotropin hormone (GtH) secretion in vivo in testosterone-implanted juvenile rainbow trout, and for the recognition of synthesized Gn-RH (s-Gn-RH) perykaria by a specific antibody raised against the s-Gn-RH in regions of the brain described as containing LH-RH immunoreactive-like material. A radioimmunoassay has been developed for the salmon Gn-RH, and its specificity to measure trout Gn-RH has been tested. Using this assay, the brain and pituitary Gn-RH contents have been measured throughout the final phases of maturation and ovulation. Brain Gn-RH increases from the end of vitellogenesis (8.9 +/- 0.76 ng/brain) to ovulation (more than 15 ng/brain). Pituitary Gn-RH is lower (1.58 +/- 0.69 ng/pituitary) at the end of vitellogenesis and follows a similar profile as in the brain, except for a significant decrease just prior the beginning of oocyte maturation. The correlations between Gn-RH levels and GtH pituitary and plasma levels show that total brain Gn-RH is never correlated to the GtH, suggesting that the increase in the brain Gn-RH content is related to a Gn-RH system closely related to maturation and ovulation, which remains to be investigated. On the contrary, pituitary Gn-RH levels are well correlated with pituitary and plasma GtH levels, indicating that pituitary Gn-RH levels might represent a good index of the Gn-RH neurosecretory activity in the fish hypothalamohypophysial complex, given the absence of a portal system in teleost.

N-Terminal domain of HTLV-I integrase. Complexation and conformational studies of the zinc finger.

The HTLV-I integrase N-terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9-43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N-terminal domain is likely to be composed of an helical region (residues 28-42) and a beta-strand (residues 20-23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region.