Dynein light chain 8a of Toxoplasma gondii , a unique conoid‐localized β‐strand‐swapped homodimer, is required for an efficient parasite growth

Abstract

Dynein light chain 8 (DLC8) is a ubiquitous eukaryotic protein regulating diverse cellular functions. We show that the obligate intracellular parasite Toxoplasma gondii harbors 4 DLC8 proteins (TgDLC8a-d), of which only TgDLC8a clusters in the mainstream LC8 class. TgDLC8b-d proteins form a divergent and alveolate-specific clade. TgDLC8b-d proteins are largely cytosolic, whereas TgDLC8a resides in the conoid at the apical end of T. gondii. The apical location of TgDLC8a is also not shared by its nearly identical Eimeria (EtDLC8a), Plasmodium (PfDLC8), or human (HsDLC8) orthologs. Notwithstanding an exclusive conoid targeting, TgDLC8a exhibits a classical LC8 structure. It forms a homodimer by swapping of the β strands that interact with the antiparallel β' strands of the opposing monomers. The TgDLC8a dimer contains two identical binding grooves and appears to be adapted for multitarget recognition. By contrast, the previously reported PfDLC8 homodimer is shaped by binding of the β strand with the parallel β' strand and lacks such a distinct binding interface. Our comparisons suggest an unexpected structural and functional divergence of the two otherwise conserved proteins from apicomplexan parasites. Finally, we demonstrate that a phosphomimetic S88E mutation renders the TgDLC8a-S88E mutant monomeric and cytosolic in T. gondii, and its overexpression inhibits the parasite growth in human fibroblasts.

Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A

“…In addition to this improved understanding of LC8, interest in this molecule has grown as LC8 has been shown to have unique and important roles in an increasing number of human pathogens. These include viruses, such as HIV (26), Ebola (27), and rabies (28), and the protistan parasite Toxoplasma gondii (29).Trypanosoma brucei, a member of the early-diverging phylogenetic order Kinetoplastida, is a vector-borne parasite that causes lethal disease in both humans and livestock (30). In T. brucei, LC8 was identified in mass spectrometry analysis of the flagellum (31) and, surprisingly, as a subunit of the class I transcription factor A (CITFA) (32, 33).…”

Molecular and Structural Characterization of the Tegumental 20.6-kDa Protein in Clonorchis sinensis as a Potential Druggable Target

“…Previous studies have indicated that the homodimeric DLCs in S. mansoni [26] and F. hepatica [27] were composed of two asymmetric monomers through β-sheet interactions. The homodimer of CsTegu20.6 was constructed using GalaxyGemini [44] with only the C chain of DLC1 (PDB ID: 4DS1_C), although the fifth β-strand (indicated with a dotted circles) as a monomeric counterpart was missed in the predicted structure [45] (Figure 5C). This result suggests that CsTegu20.6 can dimerize, confirming that this feature is conserved between the DLCs.…”

Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

“…The genome also contains annotated genes for several types of intermediate and light chains, including light chain type 1, Tctex-1, Roadblock/LC7, the axonemal assembly factor, axonemal light chain, and axonemal intermediate chain (69). Although overexpressed dynein light chain 1 (DLC1) fused to green fluorescent protein (GFP) localizes to the spindle poles, centrioles, the basal end, and the conoid region of tachyzoites (95), a subsequent study of a shortened version of the same protein (minus 49 aminoterminal residues, termed T. gondii DLC8a [TgDLC8a]) localizes it only to the apical region of tachyzoites (96). The latter study used a small epitope tag that was introduced as an in-frame fusion at the carboxy terminus of the endogenous locus.…”

“…The complement of MAPs localizing to spindle microtubules remains largely uncharacterized, though several proteins have been identified on centrioles located at the spindle poles. Since DLC1/TgDLC8a localizes to the spindle only as an overexpressed GFP fusion protein (95,96), the plus-end binding protein EB1 (J. de Leon and N. Morrissette, unpublished observations) is the sole marker of mitotic microtubules. It is quite likely that dyneins and kinesins are spindle components, in addition to other, yet-to-be-characterized MAPs.…”

“…Centrin 3 localizes to the conoid in addition to the tachyzoite centrioles (95,103,104). A dynein light chain (DLC1/DLC8a) is found in the conoid region (95,96). Toxoplasma and other simple centriole-containing eukaryotes have homologs of a SAS-6-like (SAS6L) protein (87).…”