Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 2. Three-dimensional solution structure

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D H-1 NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and mu O-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18 +/- 0.05 Angstrom for the backbone atoms and 1.39 +/- 0.33 Angstrom for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.

Three-dimensional structure of RK-1: A novel alpha-defensin peptide

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 residues, including six cysteines arranged into three disulfide bonds. It exhibits antimicrobial activity against Escherichia coli and activates Ca2+ channels in vitro. Through its physicochemical similarity, identical cysteine spacing, and linkage to the corticostatins/defensins, it was presumed to be a member of this family. However, RK-1 lacks both a large number of arginines in the primary sequence and a high overall positive charge, which are characteristic of this family of peptides. The three-dimensional solution structure, determined by NMR, consists of a triple-stranded antiparallel beta -sheet and a series of turns and is similar to the known structures of other alpha -defensins. This has enabled the definitive classification of RK-1 as a member of this family of antimicrobial peptides. Ultracentrifuge measurements confirmed that like rabbit neutrophil defensins, RK-1 is monomeric in solution, in contrast to human neutrophil defensins, which are dimeric.

Circular proteins in plants - Solution structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis

Much interest has been generated by recent reports on the discovery of circular (i.e. head-to-tail cyclized) proteins in plants. Here we report the three-dimensional structure of one of the newest such circular proteins, MCoTI-II, a novel trypsin inhibitor from Momordica cochinchinensis, a member of the Cucurbitaceae plant family. The structure consists of a small beta -sheet, several turns, and a cystine knot arrangement of the three disulfide bonds. Interestingly, the molecular topology is similar to that of the plant cyclotides (Craik, D. J., Daly, N. L., Bond, T., and Waine, C. (1999) J. Mol. Biol, 294, 1327-1336), which derive from the Rubiaceae and Violaceae plant families, have antimicrobial activities, and exemplify the cyclic cystine knot structural motif as part of their circular backbone. The sequence, biological activity, and plant family of MCoTI-II are all different from known cyclotides. However, given the structural similarity, cyclic backbone, and plant origin of MCoTI-II, we propose that MCoTI-II can be classified as a new member of the cyclotide class of proteins. The expansion of the cyclotides to include trypsin inhibitory activity and a new plant family highlights the importance and functional variability of circular proteins and the fact that they are more common than has previously been believed, Insights into the possible roles of backbone cyclization have been gained by a comparison of the structure of MCoTI-II with the homologous acyclic trypsin inhibitors CMTI-I and EETI-II from the Cucurbitaceae plant family.

Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega -Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta -hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial hits from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta -hairpin is a key site for interaction of the toxin with insect calcium channels.

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp PCC6803 DnaB mini-intein

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures similar to14 degreesC higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (DeltaDeltaG approximate to 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Structure-activity relationships of alpha-conotoxins targeting neuronal nicotinic acetylcholine receptors

alpha-Conotoxins that target the neuronal nicotinic acetylcholine receptor have a range of potential therapeutic applications and are valuable probes for examining receptor subtype selectivity. The three-dimensional structures of about half of the known neuronal specific alpha-conotoxins have now been determined and have a consensus fold containing a helical region braced by two conserved disulfide bonds. These disulfide bonds define the two-loop framework characteristic for alpha-conotoxins, CCXmCXnC, where loop 1 comprises four residues (m = 4) and loop 2 between three and seven residues (n = 3, 6 or 7). Structural studies, particularly using NMR spectroscopy have provided an insight into the role and spatial location of residues implicated in receptor binding and biological activity.

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V, hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.

The Mesozoic and Cenozoic motion of Adria (central Mediterranean): a review of constraints and limitations

This paper presents kinematic analysis on the motion of Adria, which is the continental mass that bridges Africa and Europe in the central Mediterranean. Palaeomagnetic data show a general coherence between the motion of Adria and Africa since the Late Paleozoic. This mutual motion, for the period from 120 Ma and the present, is verified by comparing inferred palaeolatitudes from relatively stable parts of Adria (Apulia, Gargano, Istria, and the Southern Alps) and the Hyblean Plateau, with latitudinal changes that are calculated from the motion of Africa with respect to hotspots. Additional constraints on the motion of Adria are provided from the Late Paleozoic-Early Mesozoic passive margin of Adria in the Ionian Sea. The seismic structure of the floor of the Ionian Sea resembles the structure of the oceanic crust in marginal back-arc basins, suggesting that it formed as a small ocean basin. Furthermore, the Ionian lithosphere in the Calabrian arc has been subjected to rapid rollback, which commonly occurs only when the subducting slab is made of oceanic lithosphere. This oceanic domain marks the Pennian-Triassic to Jurassic plate boundary between Adria and Africa, suggesting that a small amount of independent motion between Adria and Africa took place at that time. Since the Jurassic, Adria and Africa have shared a relatively coherent motion path. (C) 2004 Lavoisier SAS. All rights reserved.

Stabilization of native protein fold by intein-mediated covalent cyclization

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10degreesC by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG = 2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding I measured by stopped-flow circular dichroism. rate of less than 4 min(-1) The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E. coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding. (C) 2005 Elsevier Ltd. All rights reserved.

Inhibition of NF kappa B leads to an intracellular reducing environment in human monocyte-derived immature dendritic cells

Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

Solution structure of χ-conopeptide MrIA, a modulator of the human norepinephrine transporter

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.: Schroder, C. L; Kaye, D. M.; Adams, D. I.; Alewood, P. F.; Lewis, R. J. J Biol Client 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hypl2 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency. This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by entailing the Biopolymers editorial office at biopolymers@wiley.com (c) 2005 Wiley Periodicals, Inc.

N-methylation of diamino-substituted macrocyclic complexes: Intramolecular cyclisation

Two new macropolycyclic hexaamines L(2) and L(4) as their copper(II) complexes have been isolated as products from the condensation of the diamino-substituted macrocyclic complex trans-(6,13-dimethyl-1,4,8,11-tetraazacyclo-tetradecane-6,13-diamine)copper(II) [CuL(1)](2+) with aqueous formaldehyde. Both of the complexes exhibit methylene bridges between the pendant amine and the adjacent co-ordinated macrocyclic N-donors. Their crystal structures have been determined: [CuL(2)(NCS)][SCN], triclinic, space group P (1) over bar, a = 7.133(2), b = 9.813(2), c = 16.745(3) Angstrom, alpha = 101.05(2), beta = 99.36(2), gamma = 99.77(2)degrees, Z = 2; [CuL(4)Cl][ClO4]. H2O, triclinic, space group P (1) over bar, a = 9.3327(8), b = 10.8989(6), c = 12.672(1) Angstrom, alpha = 68.591(6), beta = 78.899(6), gamma = 87.384(6)degrees, Z = 2. The complexes exhibit square-pyramidal geometries, and significantly lower-energy electronic maxima relative to their parent complex [CuL(1)](2+). Electrochemistry of [CuL(2)](2+) revealed a reversible Cu-II-Cu-I redox couple, by contrast to those of macromonocyclic analogues.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Antibacterial activity of bovine lactoferrin-derived peptides

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified. one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf. one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond, These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ton-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC), They were characterized by. N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination, Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques, This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 mu M or less, Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC, Subfragment 1 (residues I to 10) was active against most of the test microorganisms at concentrations of 10 to 50 mu M. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 mu M. These antibacterial studies indicate that the activity of lactoferricin Is mainly, but not wholly, due to its N-terminal region.