Phallotoxins are toxic compounds produced by poisonous mushroom Amanita phalloides and belong to the class of bicyclic peptides with a transannular thioether bridge. Their intoxication mechanism in the liver involves a specific binding of the toxins to F-actin that, consequently, prevents the depolymerization equilibrium with G-actin. Even though the conformational features of phallotoxins have been worked out in solution, the exact mechanism of interaction with F-actin is still unknown. In this study a toxic phalloidin synthetic derivative, bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-Ala7)(S-3-->6) has been synthesized. A substitution at position 7. with an Ala residue replaces the 4,5-dihydroxy-Leu present in the natural phalloidin. This analogue has formed crystals suitable for X-ray analysis, and represents the first case for such a class of compounds. The solid-state structure as well as the solution conformation have been evaluated. NMR techniques have been used to extract interproton distances as restraints in subsequent molecular dynamics calculations. Finally, a direct comparison between structures in solution and in the solid state is presented.

Nodal, a member of the transforming growth factor-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. Up to date structural information on the interaction of Nodal with its molecular partners are unknown. To deepen our understanding about mechanisms underlying both embryonic development and Nodal/Cripto-dependent tumor progression, we present here a molecular model of activin receptor-like kinase 4/Cripto/Nodal complex built by homology modeling as well as docking tests aimed at identifying potential binding epitopes. Starting from this model, we have predicted a large interaction surface on Nodal, which encompasses residues 43-69 and includes the prehelix loop and the H3 helix. This hypothesis has been subsequently assessed by surface plasmon resonance binding assays between the full-length Cripto and synthetic peptides reproducing the selected Nodal regions. In addition, the binding affinity between the full-length Nodal and Cripto proteins has been evaluated for the first time.

Synthetic derivatives of phalloidin have been investigated in solution by circular dichroism (CD) and NMR spectroscopy. They differ from natural phalloidin (PHD), bicyclo(Ala 1 -d-Thr 2 -Cys 3 -cis-4-hydroxy-Pro 4 -Ala 5 -2-mercapto-Trp 6 -(OH) 2 Leu 7 )(S-3 3 6), in that they are modified at positions 2, 3, and 7. Among these synthetic analogues, structural differences and varying degrees of atropisomerism are found. By comparing the respective molecular models obtained by restrained molecular dynamics (RMD) simulations based on experimental NMR data, structural features that may be responsible for the different biological behavior become apparent. Our results indicate that the structural changes that result from an inversion of chirality of residue 3 lead to a complete loss of toxicity. Conversely, toxicity is less affected by the structural changes that stem from an inversion of chirality of residue 2. Moreover, unlike the other phallotoxins, when the thioether unit bridges to the opposite face of the main peptide ring, in contrast to the situation in other phallotoxins, large structural changes are observed as well as a total loss of activity. Molecular models of the synthetic phalloidin analogues have been used to investigate the necessary structural requirements for the interaction with F-actin. To this end, the F-actin/PHD model of M. Lorenz et al. was employed; docking experiments of our molecular models in the PHD binding site are presented.

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