The unusual dynamics of parasite actin result from isodesmic polymerization

Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here, we re-examine the polymerization properties of actin in Toxoplasma gondii (TgACTI), unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. TgACTI polymerization kinetics lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly, and the size distribution of TgACTI filaments in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers

Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility

Synthetic Chondramide A Analogues Stabilize Filamentous Actin and Block Invasion by <i>Toxoplasma gondii</i>

Apicomplexan parasites such as <i>Toxoplasma gondii</i> rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of <i>T. gondii in vitro</i>. Structural modeling of chondramide A (<b>2</b>) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (<b>2b</b>–<b>k</b>) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. <i>In vitro</i> polymerization assays using highly purified recombinant actin from <i>T. gondii</i> verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites

Nucleotide Effects on the Structure and Dynamics of Actin

Adenosine 5′-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5′-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an α-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins

Blocker Protection by Short Spermine Analogs: Refined Mapping of the Spermine Binding Site in a Kir Channel

Strongly inwardly rectifying potassium channels are blocked by intracellular polyamines with a uniquely steep voltage dependence. An understanding of the fundamental details underlying the voltage dependence of polyamine block requires a constrained structural description of the polyamine-binding site. With this goal in mind, we previously used a “blocker protection” approach to examine the effects of polyamine occupancy on the rate of MTSEA modification of cysteine residues located at pore-lining sites in a strongly rectifying Kir channel (Kir6.2[N160D]). In the study presented here, we focused this strategy to characterize the effects of polyamine analogs that are similar in size to spermine on the rate of MTSEA modification. The observed protection profile of spermine is identical to that previously reported, with spermine occupancy inhibiting MTSEA modification of residue 157C, which is deep in the Kir pore, but having little effect on modification rates of 164C or 169C, closer to the intracellular side of the inner cavity. Remarkably, slightly longer synthetic spermine analogs (BE-spermine, CGC-11098) significantly increased the protection observed at position 164C. The extended protection profile observed with slightly extended polyamine analogs significantly enhances the resolution of our previous mapping efforts using the blocker protection approach, by eliminating uncertainties regarding the blocked conformations of the much longer polyamines that were used in earlier studies. For all short polyamine analogs examined, modification at the entrance to the inner cavity (169C) was unaffected by blocker occupancy, although blocker dissociation was dramatically slowed by partial modification of this site. These data support the validity of a blocker protection approach for mapping polyamine-binding sites in a Kir pore, and confirm that spermine binds stably at a deep site in the inner cavity of strongly rectifying Kir channels