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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.

Phallotoxins form tight complexes with filamentous actm and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturmg moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments agamst shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization offilaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxm is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phallotdin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.

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