Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar.

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 Å resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.

Functional analyses of the CIF1-CIF2 complex in trypanosomes identify the structural motifs required for cytokinesis.

Cytokinesis in trypanosomes occurs uni-directionally along the longitudinal axis from the cell anterior towards the cell posterior and requires a trypanosome-specific CIF1-CIF2 protein complex. However, little is known about the contribution of the structural motifs in CIF1 and CIF2 to complex assembly and cytokinesis. Here, we demonstrate that the two zinc-finger motifs but not the coiled-coil motif in CIF1 are required for interaction with the EF-hand motifs in CIF2. We further show that localization of CIF1 depends on the coiled-coil motif and the first zinc-finger motif and that localization of CIF2 depends on the EF-hand motifs. Deletion of the coiled-coil motif and mutation of either zinc-finger motif in CIF1 disrupts cytokinesis. Furthermore, mutation of either zinc-finger motif in CIF1 mislocalizes CIF2 to the cytosol and destabilizes CIF2, whereas deletion of the coiled-coil motif in CIF1 spreads CIF2 over to the new flagellum attachment zone and stabilizes CIF2. Together, these results uncover the requirement of the coiled-coil and zinc-finger motifs for CIF1 function in cytokinesis and for CIF2 localization and stability, providing structural insights into the functional interplay between the two cytokinesis regulators.