Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Vidilaseris, Keni; Shimanovskaya, Ekaterina; Esson, Heather J.; Morriswood, Brooke; Dong, Gang

doi:10.1074/jbc.m114.554659

Cited by 15 publications

(45 citation statements)

References 41 publications

(57 reference statements)

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“…Using electron microscopy Vidilaseris et al , demonstrated that the EF-hand domains of BILBO1 change their conformation upon calcium binding, and the coiled-coil domain can form anti-parallel dimers, which can then form linear polymers via the C-terminal leucine zipper. Further, they demonstrated that these filaments can condense into fibers through lateral interactions [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

“…The primary and secondary structures of BILBO1 do not predict a specific function, and the protein does not appear to have any obvious membrane-targeting domains, but it does possess two predicted EF-hand calcium-binding domains (aa 185–213 and aa 221–249). It also has a large coiled-coil (CC) domain (aa 263–566), which is involved in protein-protein interactions [ 15 ]. Based on our hypothesis that BILBO1 is the FPC scaffold, we decided to determine 1) if BILBO1 can form polymers in vivo , 2) what domain(s) of the protein is(are) involved in polymer formation.…”

Section: Introductionmentioning

confidence: 99%

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BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

et al. 2015

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The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

et al. 2015

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“…Of these, the best characterized is an electrondense horseshoe-shaped structure named the flagellar pocket collar (FPC) (4). The only known component of the FPC is the protein TbBILBO1, which has been localized to the FPC by immunoelectron microscopy (immuno-EM) and shown to be essential for FP biogenesis (9)(10)(11)(12).…”

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confidence: 99%

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

Morriswood

Schmidt

2015

Eukaryot Cell

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The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo-and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket.T rypanosoma brucei is an important parasite of humans and domestic animals in sub-Saharan Africa, as the causative agent of sleeping sickness and nagana, respectively. Its complex life cycle involves transitions between tsetse fly vectors (its definitive hosts) and mammalian intermediate hosts. This life cycle involves a number of different cell stages, of which the procyclic form (found in the tsetse fly) and the slender bloodstream form (BSF) (found in the mammalian bloodstream) are the best studied in a laboratory setting. The procyclic form and the BSF of T. brucei share similar cytoskeletal architectures (1, 2).The principal feature of this cytoskeleton is a corset of microtubules that lie directly underneath the plasma membrane and impart to the cell its distinctive shape (3). A single invagination of the plasma membrane, termed the flagellar pocket (FP), constitutes a distinct subdomain and is found at the posterior end of the cell (4). The FP is the site of all endo-and exocytic traffic (5, 6). Abutting the FP membrane is a basal body that nucleates the single flagellum of the trypanosome cell. The flagellum exits the FP and is adhered longitudinally to the cell body along a left-handed helical path (7). Once outside the FP, the axoneme of the flagellum is paralleled by an associated intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of a paracrystalline lattice and is associated with cellular motility (8). Nucleated adjacent to the basal body is a specialized microtubule quartet that traces around the FP and then underlies the flagellum as far as the anterior end of the cell (4).The small cylinder of membrane that connects the FP to the rest of the plasma membrane constitutes a third subdomain and is called the flagellar pocket neck (FPN) (4). A number of discrete cytoskeletal structures cluster around the FPN membrane on its cytoplasmic face. Of these, the best characterized is an electrondense horseshoe-shaped structure named th...

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“…TbBILBO1 was the first reported protein component of the FPC (5). It consists of four structural domains: a globular N-terminal domain; two central EF-hand motifs; a long coiled-coil domain; a C-terminal leucine zipper (6). Analysis using electron microscopy showed that TbBILBO1 forms long filaments in vitro (6,7).…”

Section: Instroductionmentioning

confidence: 99%

“…It consists of four structural domains: a globular N-terminal domain; two central EF-hand motifs; a long coiled-coil domain; a C-terminal leucine zipper (6). Analysis using electron microscopy showed that TbBILBO1 forms long filaments in vitro (6,7). Based on its modular architecture and intrinsic property to form spiral-like structures when ectopically expressed in vivo, TbBILBO1 was proposed to act as the structural core to recruit other components during FPC assembly (8,9).…”

Section: Instroductionmentioning

confidence: 99%

Crystal structure of the TbBILBO1 N-terminal domain reveals a ubiquitin fold with a long rigid loop for the binding of its partner

Vidilaseris

Landrein

Pivovarova

et al. 2019

Preprint

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BILBO1 was the first characterized component of the flagellar pocket collar (FPC) in trypanosomes. The N-terminal domain (NTD) of BILBO1 plays an essential role in Trypanosoma 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. Mutagenesis studies suggested that another FPC protein, FPC4, interacts with TbBILBO1 via mainly contacting the three conserved aromatic residues W71, Y87 and F89 at the center of this pocket. Overall, we have determined the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design in the future.

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Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Cited by 15 publications

References 41 publications

BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

Crystal structure of the TbBILBO1 N-terminal domain reveals a ubiquitin fold with a long rigid loop for the binding of its partner

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