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

BACKGROUND: 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 P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella. RESULTS: 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. CONCLUSIONS: These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Integrated Target-Based and Phenotypic Screening Approaches for the Identification of Anti-Tubercular Agents That Bind to the Mycobacterial Adenylating Enzyme MbtA.

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene (5), that bound with high affinity to MbtA and produced a MIC90 value of 13 µm. The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS.