Structural studies of the Trypanosoma brucei protein TbBILBO1

Trypanosomes are unicellular protists in the class Kinetoplastida, an early-branching Eukaryotic lineage. Being Eukaryotes, they share many features of their cellular organisation with mammalian cells, and are increasingly used as a model system for research into fundamental biological processes. They are also obligate parasites responsible for a number of crippling diseases in both humans and livestock. African Trypanosomes (Trypanosoma brucei), which are the best understood, live in the bloodstream of an infected host. For uptake of nutrients from the host`s bloodstream and evasion of the host`s immune response, T. brucei is wholly dependent on a specialised invagination of its plasma membrane termed the flagellar pocket (FP). Biogenesis of the FP is mediated by an electron-dense cytoskeletal structure at its neck. This flagellar pocket collar (FPC) has only one known protein component, TbBILBO1. Loss of TbBILBO1 by RNAi causes a failure of FP duplication and lethality. The mechanism by which TbBILBO1 operates is currently unknown. We are proposing high-resolution structural studies of TbBILBO1 which will provide insight into its biological function in vivo and that might additionally be a future starting point for rational drug design. Specifically, we aim to determine the structure of TbBILBO1 at atomic resolution, decipher its assembly mechanism in vitro, and analyse how its assembly dynamics are mediated in vivo.

The goal of the proposed project was to elucidate three-dimensional structural information of an essential cytoskeletal protein called BILBO1 in the parasite Trypanosoma brucei. Trypanosomes are unicellular protists in the class Kinetoplastida, an early-branching Eukaryotic lineage. Being Eukaryotes, they share many features of their cellular organization with mammalian cells, and are increasingly used as a model system for research into fundamental biological processes. They are also obligate parasites responsible for a number of crippling diseases in both humans and livestock. African Trypanosomes (T. brucei), which are the best understood, live in the bloodstream of an infected host. For uptake of nutrients from the host's bloodstream and evasion of the host's immune response, T. brucei is wholly dependent on a specialized invagination of its plasma membrane termed the flagellar pocket (FP). Biogenesis of the FP is mediated by an electron-dense cytoskeletal structure at its neck. This flagellar pocket collar (FPC) so far has only one reported protein component, BILBO1. Loss of BILBO1 by RNAi causes a failure of FP duplication and lethality. The mechanism by which BILBO1 operates is currently unknown. We proposed to carry out high-resolution structural studies on T. brucei BILBO1 (TbBILBO1) so as to decipher its folding and assembly mechanism and provide insight into the potential regulation of its biological function. We expected the high-resolution structures we would obtain might additionally be a starting point for rational drug design in the future. Over the past five years, we have carried out extensive structural and functional analyses on TbBILBO1, including determination of the NMR and crystal structures of the N-terminal domain of TbBILBO1, systematic dissection of the assembly of full-length TbBILBO1 using integrative structural biology approaches, characterization of TbBILBO1 in vivo, and more recently investigation of how TbBILBO1 cooperates with other newly identified FPC proteins to fulfill its function at the FPC. Our work has greatly advanced our understanding of the architecture and assembly of TbBILBO1 and provides guidance for potential drug design targeting a surface patch of the protein as well as further characterization of the FPC in general.