Localization of the phalloidin and nucleotide-binding sites on actin.

Phalloidin was found to block nucleotide exchange in F-actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy-transfer spectroscopy. They are separated by less than 1.0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide-ATP complex from an actin digest. The peptide comprises residues 114-118, which are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815-2818]. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G-actin and it stabilizes the structure of F-actin, an event accompanied by the trapping of ADP.