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

Vidilaseris, Keni; Landrein, Nicolas; Pivovarova, Yulia; Lesigang, Johannes; Aeksiri, Niran; Robinson, Derrick R.; Bonhivers, Mélanie; Dong, Gang

doi:10.1074/jbc.ra119.010768

“…We have previously identified several key aromatic residues in BILBO1-NTD that are important for the BILBO1 function and involved in the FPC4-BILBO1 interaction (residues Y89, F89, W71, Y64) [14,15]. The crystal structure of the BILBO2-FPC4 protein complex also suggests that residues F63, F88, and W70 in the BILBO2-NTD are involved in the interaction with FPC4.…”

Section: Mutation Of Critical Interface Residues Abolishes the Bilbo2mentioning

confidence: 98%

“…This supports the hypothesis that the NTDs share a similar function as they can compensate for each other in both chimera. This is imperative because we have demonstrated that minor changes in BILBO1-NTD, such as the W71A mutant, were lethal [14].…”

Section: The N-terminal Domain Of Bilbo2 Is Homologous To the Bilbo1 mentioning

confidence: 99%

“…The reported high-resolution NMR and crystal structures of the BILBO1-NTD demonstrate that it unexpectedly adopts a ubiquitin-like fold [13,14]. The C-terminal tail of the NTD is wellfolded and rigidly wraps around the distal end of the elongated core structure.…”

Section: Introductionmentioning

confidence: 97%

“…In T. brucei, FPC4 localizes at the interface between the FPC and the HC, suggesting a role in linking the FPC-HC-MTQ structures. Static light scattering experiments demonstrated that the BILBO1-NTD and the FPC4-CTD form a stable binary complex [14].…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional studies of the first tripartite protein complex at theTrypanosoma bruceiflagellar pocket collar

Isch

¹

,

Majneri

²

,

Landrein

³

et al. 2021

Preprint

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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 the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240) by yeast two-hybrid screen, bioinformatics, functional and structural studies. We show that BILBO2 colocalizes with BILBO1 and can modulate the shape of the BILBO1 filament by interacting with the BILBO1 EF-hand domains. Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD domain 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.Author summaryTrypanosomes belong to a group of zoonotic, protist, parasites that are found in Africa, Asia, South America, and Europe and are responsible for severe human and animal diseases. They all have a common structure called the flagellar pocket (FP). In most trypanosomes, all macromolecular exchanges between the trypanosome and the environment occur via the FP. The FP is thus essential for cell viability and evading the host immune response. We have been studying the flagellar pocket collar (FPC), an enigmatic macromolecular structure at the neck of the FP, and demonstrated its essentiality for the biogenesis of the FP. We demonstrated that BILBO1 is an essential protein of the FPC that interacts with other proteins including a microtubule-binding protein FPC4.Here we identify another bona fide FPC protein, BILBO2, so named because of close similarity with BILBO1 in protein organization and functional domains. We demonstrate that BILBO1 and BILBO2 share a common N-terminal domain involved in the interaction with FPC4, and illustrate a tripartite interaction between BILBO1, BILBO2, and FPC4. Our study also provides the first atomic view of two FPC components. These data represent an additional step in deciphering the FPC structure and function in T. brucei.

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“…However, the identity and similarity reach 32% and 38% respectively between their NTD (Fig 5A). Importantly, the two residues (Y64, W71), which were previously shown to play a critical role in BILBO1's interaction with FPC4 and cell viability [9,[13][14][15], are conserved or identical in BILBO2 (i.e. F63, W70).…”

Section: The N-terminal Domain Of Bilbo2 Is Homologous To the Bilbo1 Nterminal Domainmentioning

confidence: 99%

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

Isch

¹

,

Majneri

²

,

Landrein

³

et al. 2021

Self Cite

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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.

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Intrabody induced cell death by targeting theT. bruceicytoskeletal proteinTbBILBO1

Broster-Reix

¹

,

Ramanantsalama

²

,

Primo

³

et al. 2021

Preprint

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Trypanosoma brucei belongs to a genus of protists that cause life-threatening and economically important diseases of human and animal populations in Sub-Saharan Africa. T. brucei cells are covered in surface glycoproteins some of which are used to escape the host immune system. Exo-/endocytotic trafficking of these and other molecules occurs via a single copy organelle called the flagellar pocket (FP). The FP is maintained and enclosed around the flagellum by the flagellar pocket collar (FPC). To date, the most important cytoskeletal component of the FPC is an essential, calcium-binding, polymer-forming protein called TbBILBO1. In searching for novel immune-tools to study this protein, we raised nanobodies against TbBILBO1. Nanobodies (Nb) that were selected according to their binding properties to TbBILBO1, were tested as immunofluorescence tools, and expressed as intrabodies (INb). One of them, Nb48, proved to be the most robust nanobody and intrabody. We further demonstrate that inducible, cytoplasmic expression of INb48 was lethal to these parasites, producing abnormal phenotypes resembling those of TbBILBO1 RNAi knockdown.; Our results validate the feasibility of generating functional single-domain antibody derived intrabodies to target trypanosome cytoskeleton proteins.

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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

Cited by 4 publications

References 25 publications

Structural and functional studies of the first tripartite protein complex at theTrypanosoma bruceiflagellar pocket collar

Structural and functional studies of the first tripartite protein complex at theTrypanosoma bruceiflagellar pocket collar

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

Intrabody induced cell death by targeting theT. bruceicytoskeletal proteinTbBILBO1

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