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

Abstract

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.

Keywords: BILBO1; FPC4; Trypanosoma brucei; crystal structure; crystallography; cytoskeleton; flagellar pocket collar; parasite; protein structure; protein-protein interaction; structural biology; ubiquitin; ubiquitin fold.

Figure 1.

Crystal structure of TbBILBO1-NTD. A, ribbon diagram of the TbBILBO1-NTD structure with two orthogonal views. The structure is color-ramped from blue at the N terminus to red at the C terminus. The five β strands (β1–β5) and the single α helix (α1) are labeled. B, stereo view of a part of the 2F_o − F_c map contoured at 2 σ level. C, secondary structure diagram of TbBILBO1-NTD, with the residue ranges for each structural element indicated.

Figure 2.

A long structured loop is present at the C terminus of TbBILBO1-NTD. A, superimposed 10 energy-minimized NMR conformers of TbBILBO1-NTD. Residues in the dynamic C-terminal region (residues 101–110) are listed, with the four proline residues marked by arrows. B, ribbon diagram of the crystal structure of TbBILBO1-NTD. The last 20 residues (i.e. His-96–Arg-115) comprising the loop are shown as sticks together with 2F_o − F_c electron density map around the residues contoured at 1.5 σ level. The plot was generated using CCP4 mg (24). C, ribbon diagram in the same orientation as in B and colored by temperature factors (B-factors), with blue being the most rigid and yellow the most dynamic. The average B-factor for the structure is 23 Ų. D, details of interactions between the C-terminal tail (magenta) and the TbBILBO1-NTD core (blue). The plot was generated using DIMPLOT in the LigPlot plus suite (25). Residues involved in hydrogen bond formation are shown as ball-and-stick, with oxygen, nitrogen, and carbon atoms colored in red, blue, and gray, respectively. Water molecules mediating intermolecular hydrogen bond formation are shown as cyan-colored spheres. Gray dotted lines indicate hydrogen bonds. Nonbonded residues involved in hydrophobic interactions are shown as spoked arcs.

Figure 3.

TbBILBO1-NTD has a conserved horseshoe-like surface pocket. A, crystal structure of TbBILBO1-NTD with the C-terminal loop depicted as sticks and the rest of the structure as an electrostatic surface plot. Shown on the right are two orthogonal zoom-in views of the deeply buried side chain of residue His-96 located at the beginning of the C-terminal loop. B, full electrostatic plot with the same orientation as in A to show the horseshoe-like hydrophobic pocket. C, superposition of the crystal structure (pink) onto the previously reported NMR structure (gray, PDB ID: 2MEK). The corresponding C-terminal loops in the two structures are colored in magenta and green, respectively. D, cartoon view of the crystal structure around the hydrophobic pocket of TbBILBO1-NTD, with residues in and around the pocket shown in sticks. E, electrostatic plot of the pocket with the same orientation as in D.

Figure 4.

The aromatic residues of the TbBILBO1-NTD pocket are required for TbFPC4 binding. A, cartoon view of the hydrophobic pocket of TbBILBO1-NTD with all mutated residues shown in sticks and labeled. B, ITC results of WT and mutants of TbBILBO1-NTD with TbFPC4-CTD. Mutants W71A and Y87A/F89A showed no interaction, whereas mutant Y64A showed slightly reduced binding affinity. C, SLS profiles of individual TbBILBO1-NTD (a) and TbFPC4-CTD (b), and TbFPC4-CTD mixed with WT (c) and mutants of TbBILBO1-NTD (d and e). The highest peaks in the elution profiles are normalized to the same height for easy comparison. D, purified proteins of TbBILBO1-NTD, TbFPC4-CTD, and their complex from SEC on an SDS-PAGE gel and visualized by Coomassie Blue staining. E, the same samples as in D on a native gel. F, comparison of WT and mutants of TbBILBO1-NTD alone or in complex with TbFPC4-CTD on a native gel. In contrast to the stable complex formed between the WT TbBILBO1-NTD and TbFPC4-CTD, mutants W71A and Y87A/F89A did not form complexes with TbFPC4-CTD.

Figure 5.

The aromatic residues of the TbBILBO1-NTD pocket are required for TbFPC4 recruitment in vivo. A, expression of TbBILBO1 alone in U-2 OS cells generated filamentous polymers. B, expression of TbFPC4-GFP alone showed its tight association with microtubules. C, co-expressed TbBILBO1 and TbFPC4-GFP proteins colocalized with each other. D, co-expression of TbBILBO1-Y64A and TbFPC4-GFP showed relatively robust overlap of TbFPC4 with the TbBILBO1-Y64A polymers, but some microtubule interaction is also observed (inset). E, co-expression of TbBILBO1-W71A with TbFPC4-GFP showed no colocalization of the two proteins; TbFPC4 showed microtubule pattern. Scale bars represent 10 μm, and 1 μm in the zoom images.

Figure 6.

Residue Trp-71 on TbBILBO1 is essential for its function in T. brucei. A, WB for the PCF T. brucei cells, noninduced (NI) or induced up to 96 h for the expression of Ty1-tagged TbBILBO1-WT, TbBILBO1-Y64A, TbBILBO1-W71A, or TbBILBO1-DEE/KKK. Anti-Enolase was used as a loading control. B–E, growth curves for the PCF T. brucei cells from the same experiments as in A. The error bars represent the S.E. from three independent experiments.

Figure 7.

A hypothetical model depicting the interaction between TbBILBO1-NTD and TbFPC4-CTD. TbBILBO1-NTD is shown in a gray surface plot with residues essential for TbFPC4 binding highlighted in red, and those partially affecting TbFPC4 binding colored in pink. TbFPC4 is depicted as a blue line, with regions involved in critical interactions marked as asterisks. The arrow points to the part in TbFPC4 that passes through the gap of the horseshoe-like pocket to reach the peripheral binding site outside of the pocket.