Near-atomic structure of jasplakinolide-stabilized malaria parasite F-actin reveals the structural basis of filament instability

Abstract

During their life cycle, apicomplexan parasites, such as the malaria parasite , use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and stability are temporally and spatially controlled. In contrast to canonical actin, actin 1 (Act1) does not readily polymerize into long, stable filaments. The structural basis of filament instability, which plays a pivotal role in host cell invasion, and thus infectivity, is poorly understood, largely because high-resolution structures of Act1 filaments were missing. Here, we report the near-atomic structure of jasplakinolide (JAS)-stabilizedAct1 filaments determined by electron cryomicroscopy. The general filament architecture is similar to that of mammalian F-actin. The high resolution of the structure allowed us to identify small but important differences at inter- and intrastrand contact sites, explaining the inherent instability of apicomplexan actin filaments. JAS binds at regular intervals inside the filament to three adjacent actin subunits, reinforcing filament stability by hydrophobic interactions. Our study reveals the high-resolution structure of a small molecule bound to F-actin, highlighting the potential of electron cryomicroscopy for structure-based drug design. Furthermore, our work serves as a strong foundation for understanding the structural design and evolution of actin filaments and their function in motility and host cell invasion of apicomplexan parasites.

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

“…The structure and binding site of JASP is the same in all of our different cryo-EM structures of 206 F-actin complexed with JASP Pospich et al, 2017) (Figure 6,. Its binding site overlaps to a large proportion with the one of phalloidin, as illustrated by a 208 superposition of structures ( Figure S3D-F).…”

“…First experiments using X-ray fiber diffraction, mutational studies, scanning 87 transmission electron microscopy or computational docking (Belmont et al, 1999a;Drubin et 88 al., 1993;Lorenz et al, 1993;Oda et al, 2005;Steinmetz et al, 1998), identified the binding 89 site of phalloidin at the interface of three actin protomers, but failed to determine its exact 90 position within the filament. Only recent high-resolution electron cryo microscopy (cryo-EM) 91 studies revealed the exact interaction of phalloidin and JASP with F-actin (Iwamoto et al, 2018;92 Mentes et al, 2018;Pospich et al, 2017). Both toxins bind non-covalently 93 to the same site consisting of three actin subunits from both strands and stabilize the filament 94 by extensive hydrophobic interactions.…”

“…0.00 0.00 a Pixel size after 2x binning used for processing b In parenthesis is the initial number of images or segments. c The resolution corresponds to a section of ~ 120 Å at the center of the filaments d The helical parameters were estimated from the atomic model of five consecutive subunits independently fitted to the map as described before (Pospich et al, 2017). The small variations on the helical rise are within the margin of error of the pixel size determination.…”

Electron cryomicroscopy as a powerful tool in biomedical research

“…Furthermore, we solved the structure of the non-canonical actin filaments of Plasmodium falciparum , the parasitic microorganism causing malaria, in complex with the naturally occurring cyclic peptide jasplakinolide, exemplifying the potential of cryo-EM for structure-based drug design (Fig. 2b) [55]. We identified that subtle but significant differences are responsible for the inherent instability of the filaments, which is an essential feature for proper host cell invasion of the pathogen.…”

Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and phosphate release

“…In vitro , apicomplexan actins tend to form only short filaments of ~100 nm without the filament-stabilizing macrolide jasplakinolide 7–9,11 . T. gondii actin (TgAct) has been proposed to follow an isodesmic polymerization mechanism 10 , which would differ fundamentally from the classical nucleation-elongation pathway.…”