Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo‐(<scp>L</scp>‐MeAla1‐<scp>L</scp>‐Leu2‐MePhe[(Z)Δ]3‐Gly4)], as seen by two‐dimensional 1H‐nmr spectroscopy

Pinet, Eric; Neumann, Jean‐Michel; Dahse, I.; Girault, G.; André, François

doi:10.1002/bip.360360204

Cited by 29 publications

(62 citation statements)

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“…This can be related to previous structural results obtained by NMR (30,32). Indeed, TTX and MeSer 1 -TTX exhibited the same conformation of the cyclic backbone (cis-transcis-trans configuration of the amide bond sequence) and the same interconversion among four conformers.…”

Section: Effects Of Modifications Of the Ttx Molecule On Its Inhibitorysupporting

confidence: 86%

“…Earlier NMR experiments suggested a possible self-association of TTX in aqueous solution, at 3.5 mM, at Ϫ5°C, and in the presence of salts (32). However, only a part of the data was consistent with this view: the modification of the chemical shift of the leucyl proton and the decrease of the transverse relaxation time T 2 of given backbone protons.…”

Section: Discussionmentioning

confidence: 88%

“…Florine Cavelier and Jean Verducci, Laboratoire des Amino acides, Peptides et Protéines, Université Montpellier II, Montpellier, France. The synthesis and structural properties of TTX and MeSer 1 -TTX have been reported already (30,32). Synthesis and structural features of the other derivatives will be published elsewhere.…”

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confidence: 99%

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Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Santolini¹,

Haraux²,

Sigalat³

et al. 1998

Journal of Biological Chemistry

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Eight synthetic analogues of tentoxin (cyclo-(L-N-MeGlu1 -L-Leu 2 -N-Me⌬ Z Phe 3 -Gly 4 )) modified in residues 1, 2, and 3 were checked for their ability to inhibit and reactivate the ATPase activity of the activated soluble part of chloroplast ATP synthase. The data were consistent with a model involving two binding sites of different affinities for the toxins. The occupancy of the high affinity site (or tight site) gave rise to an inactive complex, whereas filling both sites (tight ؉ loose) gave rise to a complex of variable activity, dependent on the toxin analogue. Competition experiments between tentoxin and nonreactivating analogues allowed discrimination between the absence of binding and a nonproductive binding to the site of lower affinity (or loose site). The affinity for the loose site was not affected significantly by the modifications of the tentoxin molecule, whereas the affinity for the tight site was found notably changed. Increasing the size of side chain 1 or 2 and introducing a net electrical charge both resulted in a decrease of affinity for the tight site, but the second change dominated the first one. The activity of different ternary complexes enzyme-tentoxin-analogue depended on the nature of the toxin bound on each site and not only on that bound on the loose site. This demonstrates that the reactivation process results from an interaction, direct or not, between these two binding sites. Possible molecular mechanisms are discussed.F 0 F 1 proton ATPases (or ATP synthases) are bound to energy-transducing membranes and couple the phosphorylation of ADP into ATP to the dissipation of a protonmotive force. They consist of a transmembrane proton channel (F 0 ) and an extrinsic part (F 1 ) bearing six nucleotide binding sites, catalytic and noncatalytic. The F 1 moiety is composed of five different subunits named ␣, ␤, ␥, ␦, and ⑀ (stoichiometry ␣ 9 -12]), the mitochondrial enzyme having additional subunits (5, 6). It is proposed that the F 0 moiety would work as a rotative proton-driven motor, the rotor consisting of the c subunits (7), presumably arranged in a crown (8). The rotation would be transmitted to the ␥ subunit of the F 1 moiety (9), which should modify sequentially the three catalytic sites located on ␤ subunits (4) to induce ATP synthesis (10). Experimental arguments have been presented against (11,12) and for (9, 13-15) the rotation of ␥. An essential feature of this model is that the cooperative functioning among the three catalytic sites is strictly related to the rotation of the ␥ subunit and thus to the proton pumping activity.[), produced by several phytopathogenic fungi of the Alternaria genus (16,17). Under special conditions, this toxin induces a chlorosis in some higher plants (18). It specifically inhibits ATP synthesis in isolated chloroplasts (19). In vitro and at low concentrations (10TTX inhibits the isolated chloroplast F 1 -ATPase (19 -22), but at higher concentrations (10 Ϫ5 -10 Ϫ4 M), it strongly stimulates ATPase activity (21-23). At these same concentration...

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Section: Effects Of Modifications Of the Ttx Molecule On Its Inhibitorysupporting

confidence: 86%

Section: Discussionmentioning

confidence: 88%

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Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Santolini¹,

Haraux²,

Sigalat³

et al. 1998

Journal of Biological Chemistry

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“…In aqueous solution, tentoxin exists in at least four interconverting conformers of 51% (A), 37% (B), 8% (C), and 4% (D) (29). The two major forms differ in the position of the peptide bond formed between MeAla-1 and Leu-2, which is f lipped in the B-conformer.…”

Section: Structure Of Tentoxinmentioning

confidence: 99%

Structure of spinach chloroplast F ₁ -ATPase complexed with the phytopathogenic inhibitor tentoxin

Groth

2002

Proc. Natl. Acad. Sci. U.S.A.

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Tentoxin, a natural cyclic tetrapeptide produced by phytopathogenic fungi from the Alternaria species affects the catalytic function of the chloroplast F1-ATPase in certain sensitive species of plants. In this study, we show that the uncompetitive inhibitor tentoxin binds to the ␣␤-interface of the chloroplast F1-ATPase in a cleft localized at ␤Asp-83. Most of the binding site is located on the noncatalytic ␣-subunit. The crystal structure of the tentoxininhibited CF1-complex suggests that the inhibitor is hydrogen bonded to Asp-83 in the catalytic ␤-subunit but forms hydrophobic contacts with residues Ile-63, Leu-65, Val-75, Tyr-237, Leu-238, and Met-274 in the adjacent ␣-subunit. Except for minor changes around the tentoxin-binding site, the structure of the chloroplast ␣ 3 ␤ 3 -core complex is the same as that determined with the native chloroplast ATPase. Tentoxin seems to act by inhibiting intersubunit contacts at the ␣␤-interface and by blocking the interconversion of binding sites in the catalytic mechanism.]} produced by various phytopathogenic fungi from the Alternaria species, acts as a selective energy transfer inhibitor of the chloroplast F 1 -ATPase in certain sensitive species of plants but shows no effect on the homologous mitochondrial or bacterial enzymes. In the soluble, isolated CF 1 -complex, the phytopathogen inhibits ATP hydrolysis, whereas both catalytic reactions, ATP synthesis and hydrolysis, are blocked in the membrane-bound CF 1 . Binding studies indicate that the inhibition of the ATPase by tentoxin is uncompetitive with nucleotides (1-3) and suggest that the phytopathogen prevents the cooperative release of tightly bound nucleotides from the enzyme (4). However, the precise number and location of the tentoxin-binding site(s) and the mechanism by which the phytopathogen affects the catalytic activity of the chloroplast ATPase are still unknown. Labeling studies suggest at least one high-affinity inhibitory binding site (K d Ͻ 10 Ϫ8 M), probably located at the ␣␤-interface, and 1-2 low-affinity binding sites (K d Ͼ 10 Ϫ6 M), which cause a reactivation of the enzyme (5-6). Analysis of tentoxin-sensitive and -resistant Nicotiana species indicated that residue ␤Asp-83 located at the interface of the N-terminal ␤-barrel domains in the ␣-and ␤-subunits is crucial for tentoxin binding and͞or sensitivity (7). Substitution of this residue by glutamate, alanine, or leucine caused tentoxin resistance (8), whereas substitution of the native glutamate in the tentoxin-resistant Chlamydomonas reinhardtii F 1 -ATPase by aspartate induced tentoxin sensitivity (9). On the other hand, tentoxin-resistant F 1 -ATPases from thermophilic Bacillus PS3 and from E. coli (10) contain aspartate in the equivalent position of the ␤-subunit as well. Hence, additional structural elements in the ␣-and͞or ␤-subunits are probably required for tentoxin binding and sensitivity in F 1 -ATPases. More detailed information on these critical residues was obtained from the crystal structure of the native spinach chloroplast F ...

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“…We focused on the conformational system of tentoxin, a natural cyclotetrapeptide excreted by a phytopathogenic fungus, which has been shown to possess clearly defined conformations in water, 26 an appealing feature for drug design. Its conformer distribution has been fully characterized by NMR spectroscopy at different temperatures, in various aqueous and organic solvents, 23 making it a good starting theoretical model for such an analysis.…”

Section: Introductionmentioning

confidence: 99%

Predicting the conformational states of cyclic tetrapeptides

et al. 2003

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Biologically active cyclic tetrapeptides, usually found among fungi metabolites, exhibit phytotoxic or cytostatic activities that are likely to be governed by specific conformations adopted in solution. For conformational studies and drug design, there is a strong interest in using fast and reliable methods to determine correctly the conformational population of cyclotetrapeptides. We show here that standard molecular mechanics computational approach gives satisfactory results. The method was validated step by step by experimental data either obtained after synthesis and NMR analysis, or found in the literature. The cyclo(Gly)(4), cyclo(Ala)(4), cyclo(Sar)(4), and cyclo(SarGly)(2) peptides were used to evaluate the prediction of the peptide backbone conformation, and the detailed conformational analysis of tentoxin, a natural phytotoxic cyclotetrapeptide in which N-alkylated peptide bonds alternate with regular secondary ones, was used to validate the computation of conformers proportions. From the knowledge of an initial cyclic primary structure and of the D or L configuration of the amino acids, we show that it is possible to determine the exact orientation of carbonyl groups and to predict the nature of conformers present in solution. The proportion of each conformer can be inferred from a statistical thermodynamics approach by using the potential energy values of each conformer, computed by molecular mechanics methods with the TRIPOS force field, which allowed us to account for the solvent. The solvent contribution was processed by two different methods according to the nature of the interactions: whether through the dielectric constant introduced in the electrostatic potential, when interaction with solute molecules are weak or negligible, or through the computation of free energy of solvation using the algorithm SILVERWARE for solvents explicitly interacting with the solute. When applied to tentoxin, this conformational analysis yielded results in very good agreement with the experimental data reported by Pinet et al. (Biopolymers, 1995, Vol. 36, pp. 135-152), on both the nature of existing conformers and their relative proportions, whatever the nature of the considered solvent.

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Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo‐(L‐MeAla¹‐L‐Leu²‐MePhe[(Z)Δ]³‐Gly⁴)], as seen by two‐dimensional ¹H‐nmr spectroscopy

Cited by 29 publications

References 61 publications

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Structure of spinach chloroplast F ₁ -ATPase complexed with the phytopathogenic inhibitor tentoxin

Predicting the conformational states of cyclic tetrapeptides

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Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo‐(L‐MeAla1‐L‐Leu2‐MePhe[(Z)Δ]3‐Gly4)], as seen by two‐dimensional 1H‐nmr spectroscopy

Cited by 29 publications

References 61 publications

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Interrelation between High and Low Affinity Tentoxin Binding Sites in Chloroplast F1-ATPase Revealed by Synthetic Analogues

Structure of spinach chloroplast F 1 -ATPase complexed with the phytopathogenic inhibitor tentoxin

Predicting the conformational states of cyclic tetrapeptides

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Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo‐(L‐MeAla¹‐L‐Leu²‐MePhe[(Z)Δ]³‐Gly⁴)], as seen by two‐dimensional ¹H‐nmr spectroscopy

Structure of spinach chloroplast F ₁ -ATPase complexed with the phytopathogenic inhibitor tentoxin