The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.FYVE domains are small (70 -80 amino acids) cysteine-rich domains that bind two zinc ions (1, 2). They are named for the first letters of the first four proteins in which they were identified: Fablp, YOTB, Vac1p, and early endosomal antigen 1 (EEA1).1 A functional role for the FYVE domain was first recognized when it was found to facilitate the localization of EEA1 to endosomes (3). Thereafter, it has been demonstrated that the FYVE domain specifically binds phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro (4, 5). PtdIns(3)P is constitutively present in eukaryotic cells, and the majority of PtdIns(3)P in mammalian cells is produced by class III phosphoinositide 3-kinase (6). PtdIns(3)P is thought to be involved in vesicle trafficking as mutations and inhibition of phosphoinositide 3-kinase have caused mis-sorting of vacuolar proteins, changes in vacuole morphology, and defects in the endocytic pathway (7). As expected from proposed roles of PtdIns(3)P, it has been found in specific subcellular locales, including the cytoplasmic face of early endosomes and internal vesicles of multivesicular bodies (6). Consistent with its in vitro PtdIns(3)P specificity, the FYVE domain has been also shown to be localized to these membrane sites in vivo (8, 9). Recent structural analyses of several FYVE domains (10 -13), a crystallographic structure analysis of EEA1-FYVE in particular (13), have elucidated the mechanism by which FYVE domains specifically recognize the PtdIns(3)P molecule. However, less is known about the mechanism by which FYVE domains interact with and are targeted to P...

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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.

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 alpha-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.

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.