Thesis defense of Sabrina Pospich

The proteins actin and myosin are key role players of eukaryotic life. The μm-long dynamic filaments formed by actin are a major component of the cytoskeleton. They furthermore serve as tracks for the molecular motor myosin, which powers many essential processes, such as cargo transport and muscle contraction. According to their biological relevance, malfunctions of either actin or myosin are usually linked to serious medical issues. The function of both proteins is tightly coupled to ATP hydrolysis as well as to their structure. Resolving this structure is hence key to decipher the molecular details of actin and myosin. While the overall structure of both proteins has already been resolved previously, the structural effect of ATP hydrolysis and the subsequent release of inorganic phosphate has remained elusive. Similarly, it is poorly understood how sequence variations or binding of small molecules affect the stability of actin filaments.

In my doctoral thesis I have used transmission electron cryo microscopy (cryo-EM) to solve a total of 19 high-resolution structures of filamentous actin (F-actin) as well as myosin bound to F-actin (actomyosin). First of all, I present structures of different nucleotide states, which elucidate the structural transitions of actin and myosin along their ATPase cycle. The data suggest that the ATPase cycle of myosin is not exclusively driven by mechanical coupling, but strongly relies on structural flexibility. They furthermore illustrate the conformational changes associated with ADP release and binding of ATP. The structural changes within F-actin are generally smaller than the ones in myosin, and localize primarily to the D-loop C-terminus interface at the surface of the filament. In this way, he nucleotide state is likely communicated to interaction partners, thereby facilitating the directed remodeling of the cytoskeleton. This thesis also reveals how small sequence vari-ations cause filament instability in actin 1 of the malaria parasite Plasmodium falciparum. Finally, it deciphers the structural basis by which the natural toxins jasplakinolide and phalloidin stabilize F-actin, and describes their effect on the structural transition of actin. While these small molecules already represent powerful tools for basic research, the struc-tures of a photoswitchable jasplakinolide presented in this thesis will likely promote the structure-based design of novel functionalized derivatives with even greater potential.

Collectively, the structures solved in this thesis have brought valuable insights into the structural transition of actin and myosin and thereby will eventually lead to a better understanding of all actin- and myosin-based processes.