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

Abstract

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.

Bhalin, an Essential Cytoskeleton-Associated Protein of Trypanosoma brucei Linking TbBILBO1 of the Flagellar Pocket Collar with the Hook Complex

“…We demonstrate here that BHALIN binds to BILBO1 polymers and TbBILBO1-T3 (aa 171-587) but not TbBILBO1-T4 (aa 251-587) suggesting that the TbBILBO1 EH-hand domain is required for the interaction. Two other BILBO1-binding partners interact with the EF-hand domain (the putative kinesin Tb927.7.3000 [14], and the newly identified TbBILBO2 [55] suggesting that the TbBILBO1 EF-hands play essential roles in the function of TbBILBO1, such as regulation of the interaction depending on its Ca 2+ loading status or the shape of the polymers and thus the function of the FPC.…”

TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei

“…Further, both the TbKINX1B B1BD and the BILBO1 calcium-binding EF-hands domains are necessary for the interaction. Interestingly, TbBILBO1 EF-hands are also involved in the interaction with the newly identified FPC protein TbBILBO2 [28]. However, unlike BILBO2 [21], the mutation of both EF-hands 1 and 2 of BILBO1 abolished the interaction with TbKINX1B.…”

Characterisation of TbSmee1 indicates that endocytosis is required for access of surface-bound cargo to the trypanosome flagellar pocket

“…The flagellar pocket collar is a multiprotein complex shaped like a cuff bracelet, and demarcates the boundary between the flagellar pocket and the flagellar pocket neck. It contains the protein TbBILBO1, and a number of its other components such as FPC4 and BILBO2 have recently been characterised (Albisetti et al, 2017; Bonhivers et al, 2008; Florimond et al, 2015; Isch et al, 2021). The centrin arm is a centrin-containing kinked rod which appears to be involved in the biogenesis of various cytoskeleton-associated structures (Selvapandiyan et al, 2007; Shi et al, 2008).…”