Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer

Abstract

Many human pathogens exploit the actin cytoskeleton during infection, including Toxoplasma gondii, an apicomplexan parasite related to Plasmodium, the agent of malaria. One of the most abundantly expressed proteins of T. gondii is toxofilin, a monomeric actin-binding protein (ABP) involved in invasion. Toxofilin is found in rhoptry and presents an N-terminal signal sequence, consistent with its being secreted during invasion. We report the structure of toxofilin amino acids 69 -196 in complex with the host mammalian actin. Toxofilin presents an extended conformation and interacts with an antiparallel actin dimer, in which one of the actins is related by crystal symmetry. Consistent with this observation, analytical ultracentrifugation analysis shows that toxofilin binds two actins in solution. Toxofilin folds into five consecutive helices, which form three relatively independent actin-binding sites. Helices 1 and 2 bind the symmetry-related actin molecule and cover its nucleotide-binding cleft. Helices 3-5 bind the other actin and constitute the primary actin-binding region. Helix 3 interacts in the cleft between subdomains 1 and 3, a common binding site for most ABPs. Helices 4 and 5 wrap around actin subdomain 4, and residue Gln-134 of helix 4 makes a hydrogen-bonding contact with the nucleotide in actin, both of which are unique features among ABPs. Toxofilin dramatically inhibits nucleotide exchange on two actin molecules simultaneously. This effect is linked to the formation of the antiparallel actin dimer because a construct lacking helices 1 and 2 binds only one actin and inhibits nucleotide exchange less potently.actin cytoskeleton ͉ crystal structure ͉ pathogens ͉ analytical ultracentrifugation

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

“…Actin's natural tendency to polymerize constitutes an obstacle to crystallization. Different approaches have been used to overcome this problem, including the crystallization of complexes of actin with actin-binding proteins (ABPs) (20,(22)(23)(24)(25)(26) and toxins (27), and blocking actin polymerization by mutagenesis (28) or chemical cross-linking (29). We chose to attempt Author contributions: E.D.K.…”

Host cell manipulation by the human pathogen Toxoplasma gondii

“…This protein caps microfilaments and binds globular actin, making it unavailable for microfilament (re)polymerization [51]. The newly available crystal structure [52] showing that toxofilin uses several α-helices to embrace a host actin dimer should be invaluable for designing mutants to test its role in cortical cytoskeleton traversal.…”

Toxofilin upregulates the host cortical actin cytoskeleton dynamics facilitating Toxoplasma invasion

“…n$18 cells for each condition, with a minimum of 450 measurements per condition. exposed in the actin filament (Lee et al, 2007). Because the highly conserved Ras-GAP family member Aip1 has a binding site that has been mapped to this cleft and cooperates with cofilin to regulate actin turnover (Rodal et al, 1999;Ono, 2003), we checked whether toxofilin could similarly act by cooperating with cofilin/actin depolymerizing factor (ADF) in the host cells.…”

“…Analysis of the atomic structure of the toxofilin -G-actin complex revealed that toxofilin, which is made up of five helices, binds to the actin nucleotide at residue Gln134 in helix 4, and that one toxofilin molecule interacts with two actin molecules organized as an antiparallel dimer. Although toxofilin appears to be a potent actin nucleotide-exchange inhibitor (Lee et al, 2007), its function in vivo remains undefined.…”