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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Structure of the C. Elegans ZYG-1 Cryptic Polo Box Suggests a Conserved Mechanism for Centriolar Docking of Plk4 Kinases

Plk4 family kinases control centriole assembly. Plk4s target mother centrioles through an interaction between their cryptic polo box (CPB) and acidic regions in the centriolar receptors SPD-2/Cep192 and/or Asterless/Cep152. Here, we report a crystal structure for the CPB of C. elegans ZYG-1, which forms a Z-shaped dimer containing an intermolecular β sheet with an extended basic surface patch. Biochemical and in vivo analysis revealed that electrostatic interactions dock the ZYG-1 CPB basic patch onto the SPD-2-derived acidic region to promote ZYG-1 targeting and new centriole assembly. Analysis of a different crystal form of the Drosophila Plk4 (DmPlk4) CPB suggests that it also forms a Z-shaped dimer. Comparison of the ZYG-1 and DmPlk4 CPBs revealed structural changes in the ZYG-1 CPB that confer selectivity for binding SPD-2 over Asterless-derived acidic regions. Overall, our findings suggest a conserved mechanism for centriolar docking of Plk4 homologs that initiate daughter centriole assembly

Interaction between the flagellar pocket collar and the hook complex <i>via</i> a novel microtubule-binding protein in <i>Trypanosoma brucei</i>

<div><p><i>Trypanosoma brucei</i> belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP) but remains attached to the cell body <i>via</i> the flagellum attachment zone (FAZ). The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ) that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC) circumvents the flagellum. Overlapping the FPC is the hook complex (HC) (a sub-structure of the previously named bilobe) that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of <i>in vitro</i> and <i>in vivo</i> approaches to identify and characterize a new BILBO1 partner protein—FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.</p></div

FPC4 binds to the remnant MtQ complex of isolated flagella.

<p>(A) Co-labelling of myc-tagged proteins (green) and tubulin (red) on isolated flagella from WT cells (a), and cells over-expressing myc-FPC4 (b) or myc-FPC4-ΔB1BD (c). White arrowheads are pointing to the zoom areas. Scale bars 5 μm. (B) Immuno-gold labelling of myc-FPC4-ΔB1BD (anti-myc, 10 nm gold beads) and tubulin (15 nm gold beads) on isolated flagella. In the zoom image (b), the arrows highlight the MtQ decorated with anti-tubulin and anti-myc labelling that appears close to the MtQ. In the zoom image (c), the black arrowheads highlight the FPC structure decorated by the anti-myc antibody. All the experiments were performed more than three independent times.</p

The B1BD of FPC4 is not involved in the targeting to the FPC/hook complex.

<p>(A) Immunofluorescence on cell lines over-expressing (48h of induction) myc-FPC4 (a), myc-FPC4-ΔB1BD (b), myc-FPC4-B1BD (c, d), and GFP-FPC4 (24 h of induction) (e) using anti-myc or anti-GFP (green) and anti-BILBO1 (red). Immunofluorescence was done on cytoskeletons except in (d) were cells were permeabilised. Kinetoplasts and nuclei were DAPI stained. Scale bars represent 5 μm. Initial immunofluorescence analyses were performed on two individual clones. (B) Growth curves for WT cells (grey diamond), and cells non-induced (black square) or induced (white square) for the expression of myc-FPC4, myc-FPC4-ΔB1BD, myc-FPC4-B1BD, and GFP-FPC4. Error bars represent the standard error from 3 independent experiments (errors bars are smaller than the data point mark). (C) Western blot analysis of the level of expression of myc-FPC4, myc-FPC4-ΔB1BD, and GFP-FPC4 using anti-FPC4, and <i>Tb</i>SAXO (mAb25) as loading control. Whole cell extracts from 5.10⁶ cells were loaded on 12% SDS-PAGE gels and immuno-blotted.</p

BILBO1—FPC4 interaction involves their N-terminal and C-terminal domains respectively.

<p>(A) BILBO1-FPC4 yeast two-hybrid interaction test. Left panel: Schematic overview of BILBO1 and FPC4 domains and of the combinations tested by Y2H. The EF-hand calcium binding sites of BILBO1 are represented in green and the coiled-coil domains in BILBO1 and FPC4 are represented in blue. The FPC4 B1BD is represented in violet. Right panel: The interactions tested were probed on–histidine selective medium (-His) and on growth control medium (control) (n = 3). Positive control involved p53 and T-antigen, whereas negative control involved Lamin and T-antigen. (B) Heterologous expression and co-expression in U-2 OS cells of FPC4 and FPC4 truncations fused to a C-terminal GFP tag, and of BILBO1. BILBO1 (a), FPC4 (b), FPC4-ΔB1BD (c) and FPC4-B1BD (d) were expressed alone and cells were probed with anti-BILBO1 (red) and anti-GFP (green). In e, f, g, cells were expressing BILBO1 + FPC4, BILBO1 + FPC4-ΔB1BD, and BILBO1 + FPC4-B1BD respectively and were also probed with anti-BILBO1 (red) and anti-GFP (green). Cells were extracted before labelling, except in (d) to show the cytoplasmic localisation of FPC4-B1BD, which is not visible on extracted cells. The transfections were performed more than three independent times. Nuclei were DAPI stained (blue). Scale bar represents 10 μm.</p