Crystal structure of the N-terminal domain of the trypanosome flagellar protein BILBO1 reveals a ubiquitin fold with a long structured loop for protein binding

Trypanosoma brucei is a protist parasite causing sleeping sickness and nagana in sub-Saharan Africa. T. brucei has a single flagellum whose base contains a bulblike invagination of the plasma membrane called the flagellar pocket (FP). Around the neck of the FP on its cytoplasmic face is a structure called the flagellar pocket collar (FPC), which is essential for FP biogenesis. BILBO1 was the first characterized component of the FPC in trypanosomes. BILBO1's N-terminal domain (NTD) plays an essential role in T. brucei FPC biogenesis and is thus vital for the parasite's survival. Here, we report a 1.6-Å resolution crystal structure of TbBILBO1-NTD, which revealed a conserved horseshoe-like hydrophobic pocket formed by an unusually long loop. Results from mutagenesis experiments suggested that another FPC protein, FPC4, interacts with TbBILBO1 by mainly contacting its three conserved aromatic residues Trp-71, Tyr-87, and Phe-89 at the center of this pocket. Our findings disclose the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design targeting BILBO1 to combat T. brucei infections.Alliance française contre les maladies parasitaire

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 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.Pourquoi et comment les trypanosomes construisent un Collier de la Poche FlagellaireAlliance française contre les maladies parasitaire

Structural studies of the shortest extended synaptotagmin with only two C2 domains from Trypanosoma brucei

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