Cryo‐EM Resolves Molecular Recognition Of An Optojasp Photoswitch Bound To Actin Filaments In Both Switch States

Abstract

Actin is essential for key processes in all eukaryotic cells. Cellpermeable optojasps provide spatiotemporal control of the actin cytoskeleton, confining toxicity and potentially rendering F-actin druggable by photopharmacology. Here, we report cryo electron microscopy (cryo-EM) structures of both isomeric states of one optojasp bound to actin filaments. The high-resolution structures reveal for the first time the pronounced effects of photoswitching a functionalized azobenzene. By characterizing the optojasp binding site and identifying conformational changes within F-actin that depend on the optojasp isomeric state, we refine determinants for the design of functional F-actin photoswitches. Actin is a key player in eukaryotic cell biology and involved in cellular motility, cytokinesis, intra-cellular cargo transport and endocytosis. [1] The 42 kDa globular protein (G-actin) assembles into polar filaments (F-actin) that form complex and dynamic networks. To fulfill its diverse functions, the conformation and dynamics of actin filaments are clocked by ATP hydrolysis and the subsequent release of phosphate. [2, 3] Furthermore, actin polymerization and the assembly of higher order structures is tightly controlled in space and time by a plethora of actin binding proteins (ABPs). [4] While actin may serve as a potential entry for drug design due to its involvement in many cellular processes, it is also

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

“…We collected cryo-EM data sets of the different samples (Table 1) and processed them using the helical processing pipeline implemented in the SPHIRE package (Moriya et al, 2017; Pospich et al, 2021; Stabrin, Schoenfeld, Wagner, Pospich, Gatsogiannis, & Raunser, 2020a), which applies helical restraints but no symmetry. For each data set, two all-particle density maps were reconstructed (Table 1—Supplementary Figure 1, see Methods for details).…”

“…Instead, the software offers the usage of constraints tailored to helical specimen, e.g. on the tilt angle and shift along the filament, to guide the refinement (also see Methods of (Pospich et al, 2021)). For all 3D refinements the tilt angle was softly restrained to the equator during exhaustive searches (--theta_min 90 --theta_max 90 --howmany 10).…”

Discovery of an F-actin–binding small molecule serving as a fluorescent probe and a scaffold for functional probes

“…Very recently, two novel techniques for optical manipulation of F-actin have been reported using optojasps ( 28 , 29 ), a conjugate between jasplakinolide and photoswitchable azobenzene, and Nvoc-CytoD ( 30 ), a photocaged cytochalasin D, which directly affect F-actin. While the inhibitory action of those drugs has been precisely defined, the mechanism underlying GLIFin-mediated actin perturbation remains to be established.…”

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

“…We collected cryo-EM data sets of the different samples (Table 1) and processed them using the helical processing pipeline implemented in the SPHIRE package (Moriya et al, 2017;Pospich et al, 2021;Stabrin, Schoenfeld, Wagner, Pospich, Gatsogiannis, & Raunser, 2020a), which applies helical restraints but no symmetry. For each data set, two allparticle density maps were reconstructed (Figure 1-figure supplement 1, see Methods for details).…”