Search

In an attempt to increase the antimicrobial activity of the insect defensin from Anopheles gambiae, which is active against Staphylococcus aureus at low concentration, hybrid defensins were designed by combining conserved sequence regions and variable regions of insect defensins. Their activity against S. aureus strains sensitive and resistant to conventional antibiotics was evaluated, and the toxicity of the most active molecules was tested. The three-dimensional structure of Anopheles gambiae defensin and five hybrids were determined by NMR and molecular modelling. This strategy led to the design of two chimeric defensins with increased activity compared with the native molecule, but one of them appears to be toxic to mice at a rather low concentration. The structure of the CS alphabeta motif, which is a characteristic of insect defensin, is sensitive to sequence modifications, in particular in the N-terminal loop. The existence of the CS alphabeta is most probably a prerequisite for the stability and the activity of the molecule, but is not sufficient by itself since the hybrid displaying the best defined structure is not active against the tested strains. The analysis of the structure, in relation with the activity and the toxicity data, underlines the importance of turns and of the N-terminal loop. Residues located in the turns contributing to the preservation of positive electrostatic areas at the surface of the molecules seem particularly important for the activity of the molecule, while residues involved in the N-terminal loop are both involved in the modulation of the activity and the toxicity of the molecule., (2008 Wiley-Liss, Inc.)

Virtually all cancer biological attributes are heterogeneous. Because of this, it is currently difficult to reconcile results of cancer transcriptome and proteome experiments. It is also established that cancer somatic mutations arise at rates higher than suspected, but yet are insufficient to explain all cancer cell heterogeneity. We have analyzed sequence variations of 17 abundantly expressed genes in a large set of human ESTs originating from either normal or cancer samples. We show that cancer ESTs have greater variations than normal ESTs for >70% of the tested genes. These variations cannot be explained by known and putative SNPs. Furthermore, cancer EST variations were not random, but were determined by the composition of the substituted base (b0) as well as that of the bases located upstream (up to b - 4) and downstream (up to b + 3) of the substitution event. The replacement base was also not randomly selected but corresponded in most cases (73%) to a repetition of b - 1 or of b + 1. Base substitutions follow a specific pattern of affected bases: A and T substitutions were preferentially observed in cancer ESTs. In contrast, cancer somatic mutations [Sjoblom T, et al. (2006) Science 314:268-274] and SNPs identified in the genes of the current study occurred preferentially with C and G. On the basis of these observations, we developed a working hypothesis that cancer EST heterogeneity results primarily from increased transcription infidelity.

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.

Insect peptides are key elements of the innate immunity against bacteria and fungi. These molecules offer remarkable properties: high efficacy, a low probability of resistance, limited toxicity, and immunogenicity. In this context, we are investigating several classes of peptides, and we have been successful in identifying biologically important classes of peptides and small molecules that will provide a stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and other pathologies of high medical need. Recently, we have isolated a new class of antifungal peptides from the coleopteran Acrocinus longimanus. Three homologous peptides, Alo-1, Alo-2, and Alo-3, with sequence identity above 80% and active against the Candida glabrata yeast strain were identified. Alo-3 displayed the highest activity against Candida glabrata and was thus chosen for structure determination using NMR spectroscopy and molecular modeling. Alo-3 contains six cysteine residues forming three disulfide bridges. The pairing of the cysteines was assessed using ambiguous disulfide restraints within the ARIA software, allowing us to establish that Alo-3 belongs to the inhibitor cystine-knot family. It exhibits all the structural features characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first strand to the second strand and a series of turns. To our knowledge, Alo-3 is the first peptide from insects with antimicrobial activity adopting the knottin fold. Alo-3 shows a level of activity significantly higher against C. glabrata than Alo-1 or Alo-2. It has no negatively charged residues and displays on its surface a cationic pole that may account for its antifungal activity. This finding is validated by the comparison of the structure of Alo-3 with the structure of other structurally related peptides from other sources also showing antifungal activity.

The cytotoxic ribonuclease alpha-sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of alpha-sarcin through measurement of longitudinal relaxation rates R1, off resonance rotating frame relaxation rates R1 rho, and the 15N[1H] NOE of the backbone amide 15N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. alpha-Sarcin behaves as an axial symmetric rotor of the prolate type (D(axially)/D(radially)=1.16 +/- 0.02) which tumbles with a correlation time tau(m) of 7.54 +/- 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that alpha-sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the mu s to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of alpha-sarcin in the process of recognition of its ribosomal target.

Camelids, (dromedaries, camels, and llamas) produce heavy-chains antibodies, with their antigen recognition sites composed of a single VH-like domain, referred to as VHH. The solution structure of one of these VHHs domains (VHH-H14), raised against the alpha subunit of the human chorionic gonadotropin hormone (hCG), has been determined by (15)N heteronuclear three-dimensional NMR spectroscopy. The framework is well resolved within the set of 20 best-calculated NMR structures and is close to that of classical VH domains from vertebrate antibodies, consisting of two antiparallel beta-sheets organized in a beta-barrel. Loops display a lower precision, especially the Complementarity Determining Regions (CDRs), involved in antigen recognition. Comparison of the three-dimensional VHH-H14 solution structure with its previously solved crystal structure (Spinelli et al., Nature Struct. Biol. 1996;3:752-757) reveals a high similarity to the framework, whereas significant conformational differences occur on CDRs, leading to the assumption that the antigen recognition site is a more mobile part. In order to deepen our insights into the dynamics of VHH-H14 in solution, (15)N relaxation was measured with longitudinal R1 and transverse R2 self-relaxation rates, and (15)N steady-state heteronuclear nuclear Overhauser enhancements (NOE), making it possible to probe picosecond-to-millisecond internal motions. Determination of dynamic parameters (S(2), tau(e), and Rex) through the Lipari-Szabo Model-free approach enables the identification of several regions with enhanced dynamics. Especially, the mobility measurements from NMR confirm that the antigen recognition site is the most mobile part of the VHH-H14 domain on picosecond-to-nanosecond fast time scales. Several residues belonging to the three CDRs are submitted to chemical exchange processes occurring on slow microsecond-to-millisecond time scales, suggesting that the formation of the VHH/antigen complex should be accompanied by structural changes., (Copyright 2002 Wiley-Liss, Inc.)

The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein chymotrypsin inhibitor 2. Dihedral angle restraints for the phi and psi angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond 3hbJNC' coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

By transferring the central curaremimetic beta hairpin of the snake toxin alpha into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three-dimensional structure and partially reproduced the function of the parent beta hairpin, without perturbing the three-dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin alpha, in order to examine the beta hairpin motions in both structural contexts. Thus, 13C R1, R1rho and 1H-->13C nOe were measured for all the CalphaHalpha and threonine CbetaHbeta vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified beta sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its beta structure is less stable on a minute to hour time scale. On the other hand, when the beta hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin alpha, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the beta hairpin sequence, and the beta sheet seems more stable in toxin alpha than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another.

The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.

The three-dimensional structure of the fMet-tRNA(fMet) -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu small middle dotaminoacyl-tRNA small middle dot GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNA(fMet)-binding site of IF2.

The (15)N steady-state magnetization in the presence of off-resonance rf irradiation is an analytical function of the T(1)/T(2) ratio and of the angle between the (15)N effective field axis and the static magnetic field direction. This relation holds whatever the relaxation mechanisms due to motions on the nanosecond time scale, and the size of the spin system. If motions on the micro- to millisecond time scale are present (fast exchange), the same observable depends also on their spectral density at the frequency of the effective field. The cross-peak intensity in each 2D (15)N-(1)H correlation map is directly related to the dynamic parameters, so that the characterization of fast exchange phenomena by this method is in principle less time-consuming than the separate measurement of self-relaxation rates. The theory of this approach is described. Its practical validity is experimentally evaluated on a (15)N-labeled 61 amino acid neurotoxin. It turns out that existing equipments lead to non-negligible biases. Their consequences for the accuracy attainable, at present, by this method are investigated in detail.

Motions of the backbone C alpha H alpha and threonine C beta H beta bonds of toxin alpha were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H-->13C NOE were obtained, as well as the variations of R1 rho (90 degrees) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097-16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several C alpha H alpha and threonine C beta H beta experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin alpha, a highly stable protein (Tm = 75 degrees C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1-0.5 ps, to 10-100 ps, 1 ns, and about 30 microseconds to 10 ms.

Calcicludine, a 60-amino acid protein isolated from the green mamba venom, has been recently identified as blocking a large set (i.e., L-, N- and P-type) of Ca2+ channels. The three-dimensional structure of calcicludine has been determined by NMR and molecular modeling using a data set of 723 unambiguous and 265 ambiguous distance restraints, as 33 phi and 13 chi1 dihedral angle restraints. Analysis of the 15 final structures (backbone root-mean-square deviation = 0.6 A) shows that calcicludine adopts the Kunitz-type protease inhibitor fold. Its three-dimensional structure is similar to that of snake K+ channel blockers dendrotoxins. Conformational differences with protease inhibitors and dendrotoxins are localized in the 3(10) helix and loop 1 (segments 1-7 and 10-19), the extremity of the beta-hairpin (segment 27-30), and loop 2 (segment 39-44). These regions correspond to the functional sites of bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxins. The positioning of the N-terminal segment 1-7 relative to the rest of the protein is characteristic of calcicludine. The involvement of this segment and the positively charged K31 at the tip of the beta-hairpin in the biological activity of calcicludine is discussed.

We describe a method allowing the determination of the effective B1 field amplitude distribution in a high-resolution NMR spectrometer. This method which can be adapted to almost any sequence, essentially consists of a mutation followed by a purging B0 gradient pulse. Experimental results obtained with this approach are described in homonuclear and heteronuclear cases. The experimental distributions are used to estimate the biases induced by B1 inhomogeneity, as well as the loss of RF power on heteronuclear transverse self-relaxation rate determination. In this type of measurement, the experimental biases induced on the intensities can be as large as 5% for long mixing times., (Copyright 1999 Academic Press.)

kappa-Conotoxin PVIIA from the venom of Conus purpurascens is the first cone snail toxin that was described to block potassium channels. We synthesized chemically this toxin and showed that its disulfide bridge pattern is similar to those of omega- and delta-conotoxins. kappa-conotoxin competes with radioactive alpha-dendrotoxin for binding to rat brain synaptosomes, confirming its capacity to bind to potassium channels; however, it behaves as a weak competitor. The three-dimensional structure of kappa-conotoxin PVIIA, as elucidated by NMR spectroscopy and molecular modeling, comprises two large parallel loops stabilized by a triple-stranded antiparallel beta-sheet and three disulfide bridges. The overall fold of kappa-conotoxin is similar to that of calcium channel-blocking omega-conotoxins but differs from those of potassium channel-blocking toxins from sea anemones, scorpions, and snakes. Local topographies of kappa-conotoxin PVIIA that might account for its capacity to recognize Kv1-type potassium channels are discussed.

Toxin alpha from Naja nigricollis (61 amino acids, four disulfide bridges) belongs to the "three finger" fold family, which contains snake toxins with various biological activities and nontoxic proteins from different origins. In this paper, we report an extensive 1H and 15N NMR study of the dynamics of toxin alpha in solution. 15N relaxation, 1H off-resonance ROESY, and H-D exchange experiments allowed us to probe picosecond to hour motions in the protein. Analysis of these NMR measurements demonstrates that toxin alpha exhibits various time scale motions, i.e., particularly large amplitude picosecond to nanosecond motions at the tips of the loops, observable microsecond to millisecond motions around two disulfide bridges, second time scale motions around the C-N bonds of asparagine and glutamine side chains which are more or less rapid depending on their amino acid solvent accessibility, and minute to hour motions in the beta-sheet structure. The less well-defined regions of toxin alpha solution structures are subject to important picosecond to nanosecond motions. The toxic site is organized around residues belonging to the rigid core of the molecule but also comprises residues exhibiting dynamics on various time scales. The Malpha1 epitope is subject to large picosecond to millisecond motions, which are probably modified by the interaction with the antibody. This phenomenon could be linked to the neutralizing properties of the antibody.

The advantages of using off-resonance rf fields in heteronuclear self-relaxation experiments are explored on a fully (15)N-enriched protein. It is firstly shown that in the absence of slow motions the longitudinal and transverse (15)N self-relaxation rate values derived with this method are in agreement with the ones measured by the classical inversion-recovery and Carr-Purcell-Meiboom-Gill (CPMG) sequences, respectively. Secondly, by comparing the (15)N transverse self-relaxation rates obtained by the proposed off-resonance sequence and by the CPMG sequence, 11 residues out of the 61 of toxin α are shown to exhibit a chemical exchange phenomenon in water on a time scale ranging from 1 µs to 100 ms. By varying the effective field amplitude, chemical exchange processes involving these residues are measured and the corresponding correlation times are evaluated without having assumed any motion model. Similar, though less precise, results are given by the analysis of the (15)N off-resonance self-relaxation rates on the basis of the Lipari-Szabo model to describe the fast internal dynamics of toxin α.

The alpha/beta scorpion fold consisting of a short alpha-helix and beta-sheet is a structural motif common to scorpion toxins, insect defensins, and plant gamma-thionins that invariably contains three disulfides. CHABII is a two-disulfide derivative of the scorpion toxin charybdotoxin (ChTX), chemically synthesized by inserting two L-alpha-aminobutyric acids in place of the two half-cystine residues involved in the disulfide 13-33. This disulfide is one of the two disulfides which connect the alpha-helix to the beta-sheet. The solution structure of CHABII was determined at pH 6.3 and 5 degrees C using 2D NMR and simulated annealing from 513 distance and 46 dihedral angle constraints. The NMR structure of CHABII is well-defined as judged from the low value of the averaged backbone rms deviation between the 30 lowest energy structures and the energy-minimized mean structure ((rmsd) = 0.65 A for the entire sequence and 0.48 A for the segment 3-36). Analysis and comparison of the solution structures of CHABII and ChTX lead to the following conclusions: (i) the fold of CHABII is similar to that of ChTX as indicated by the low value of the averaged backbone atomic rms deviation between the 10 lowest energy solution structures of the two proteins (1.44 A); (ii) the packing of the hydrophobic core is well-preserved, underlying the critical structural role of the hydrophobic interactions even for such a small and cysteine-rich protein as ChTX.

This brief review is devoted to the presentation of the major toxic proteins found in venoms of animals from five phyla. It is shown that various groups of venomous animals, including scorpions and snakes, produce toxins that exert different functions although they adopt a similar structure, suggesting that these toxins result from a divergent evolution. On the opposite, it is shown that toxins produced by animals from different phyla can exert similar functions even though they adopt unrelated structures, suggesting a convergent evolution. Finally, this review describes, at the molecular level, the functional and structural properties, including the dynamical characteristics, of a snake toxin which binds to the peripheral nicotinic acetylcholine receptor.

The alpha/beta scorpion fold is shared by scorpion toxins, insect defensins, and plant thionins. This small and functionally versatile template contains an alpha-helix and a triple beta-sheet linked by three disulfide bridges. With the view to introduce novel functional centers within this fold, we replaced the sequence (the cysteines and glycines excepted) of the original beta-hairpin of a scorpion toxin by the sequence of a beta-hairpin that forms part of the site by which snake neurotoxins bind to nicotinic acetylcholine receptors (AcChOR). The resulting chimeric protein, synthesized by chemical means, binds to AcChOR, though with a lower affinity than the snake toxins [Drakopoulou; E., Zinn-Justin, S., Guenneugues, M., Gilquin, B., Ménez, A., & Vita, C. (1996) J. Biol. Chem. 271, 11979-11987]. The work described in this paper is an attempt to clarify the structural consequences associated with the transfer of the beta-hairpin. We report the determination of the three-dimensional solution structure of the chimeric protein by proton NMR spectroscopy and molecular dynamics calculations. Comparison of the structure of the chimera with those of the scorpion alpha/beta toxin and of the snake neurotoxin shows that (i) the new protein folds as an alpha/beta motif and (ii) the beta-hairpins of the chimera and of the curaremimetic toxin adopt a similar conformation. A closer inspection of the differences between the structures of the original and transferred beta-hairpins allows rationalization of the biological properties of the chimera.