Amanitin is a bicyclic octapeptide and a potent inhibitor of RNA polymerase II, with extremely high affinity for the transcribing complex (K d ΒΌ 10 Γ9 ). Phalloidin is a bicyclic heptapeptide with a similar affinity for F-actin. Both of these peptides contain the characteristic tryptathionine bridge; phalloidin just having the simple thioether, and a-amanitin possessing an oxidized form of the same bridge (where the sulfur has been stereoselectively oxidized to the (R)-sulfoxide). Given this general bicyclic scaffold, there are many other derivatives of these compounds found in nature, and these are summarized in Figure 2.
Both bicyclic peptides target large proteins. Actin is a filamentous structural protein without enzymatic activity that is targeted by phalloidin. Phalloidin binds to F-actin and stabilizes its filamentous form to prevent its depolymerization into G-actin, such that the cell can no longer operate. RNA polymerase II is a 10-subunit megadalton enzyme that is targeted by amanitin. Amanitin binds not to the active site of RNA polymerase II, but to a protein fold at a nearby allosteric site, resulting in inhibition of transcription.
Despite many years of study, the side chains and exact conformational aspects responsible for the extremely high affinities of these peptides to their protein targets are still not fully understood. As F-actin is a polymer, there is no high resolution structure of phalloidin bound to the F-actin. However, X-ray powder diffraction studies have provided a structure of phalloidin bound to F-actin at the interface of two actin subunits at 8-A Λresolution. 2,3 Recent studies on a cocrystal structure of RNA pol II and a-amanitin at 2.8-A resolution revealed good evidence for a strong hydrogen bond between the 4-hydroxyproline and Glu822 of the enzyme. Also of importance were the backbone contacts