A doublecortin-domain protein of Toxoplasma and its orthologues bind to and modify the structure and organization of tubulin polymers

Abstract

TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma. TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25-alpha and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella. We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a "C"-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or "C"-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite. These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

“…falciparum apical annuli proteins. Together with recent reports on conserved proteins of the conoid structure (Bertiaux et al, 2021; Koreny et al, 2021; Leung et al, 2020; Wall et al, 2016) the presence of apical annuli in P . falciparum may indicate a similar and conserved organisation of the apical pole in Apicomplexan parasites.…”

Pseudo‐repeats in doublecortin make distinct mechanistic contributions to microtubule regulation

“…In this context, different domains within MAPs effectively act as stage‐specific tubulin chaperones specializing in interactions with different tubulin conformations. This is evident even in evolutionarily distant organisms, such as, for example, Toxoplasma gondii, where a DC domain containing protein TgDCX is essential to induce curvature in tubulin polymers (Leung et al , 2020). The presence of DC domain pseudo‐repeats with different functional specializations supports multiple activities in DCX, including acceleration of MT nucleation, control of MT architecture, MT stabilization, and interaction with other components of MT cytoskeleton (Liu et al , 2012), all within a single molecule.…”

Cryo-ET of Toxoplasma parasites gives subnanometer insight into tubulin-based structures

“…To accomplish this, they deploy a sophisticated and complex strategy involving a specialized, apical architecture and tightly choreographed secretion from two distinct secretory organelles: micronemes and rhoptries (2)(3)(4). Among the events associated with invasion is rearrangement of the cytoskeleton at the apical end, including protrusion of the conoid, a prominent bundle of spirally organized fibers (5)(6)(7)(8).…”