AbstractDespite much being known about tubulin, there is a dearth of structural information on proteins that are involved folding, processing and manipulating this important component of the cytoskeleton. Trypanosomatids can be used to investigate these proteins, as they are primitive eukaryotes whose main cytoskeletal component are microtubules. It was envisaged that conserved features discovered would elucidate basic features of eukaryotic biology and specific differences would help to understand the specialisation of these parasites. Therefore several projects were initiated in order to further understanding of several microtubule modifying and manipulation proteins in trypanosomatids. This resulted in two X-ray crystallography structures being produced. One is of the ADP ribosylation factor like 1 (ARL1) and another of the CAP-Gly domain of tubulin binding cofactor B (TBC-B). Enzymes involved in posttranslational modifications of tubulin, tyrosine ligation and deacetylation, were also investigated. These projects did not result in crystal structures, but are discussed in chapters 5 and 6.In chapter 3, the structure of Leishmania major ADP ribosylation factor-like 1 (LmARL1) is described. ARL proteins are a family of small regulatory GTPases that undergo conformational changes upon nucleotide binding. Such changes are implicated in regulating the affinity of ARLs for binding other proteins, lipids or membranes. Much is known about the structure of human and yeast ARL proteins, research stimulated in large part due to their essential nature in vesicular biogenesis and membrane trafficking. There is however, a paucity of structural data on this family of proteins in the Kinetoplastida, despite biological studies implicating them in key events related to vesicular transport and regulation of microtubule dependent processes. The crystal structure of ARL1 from the protozoan parasite L. major in complex with GDP was determined to 2.1 Å resolution. The analysis reveals a high degree of structural conservation with human ADP ribosylation factor 1 (ARF1). Mining L. major genomic data identified putative ARF/ARL family members, which were examined and classified, based on conservation of amino acid sequences combined with what is known about orthologous proteins in Kinetoplastids and in humans, for which a standardised nomenclature has been proposed. This initial classification may guide future studies designed to elucidate biological function of specific family members. Chapter 4 contains the discussion of the two functional domains of Trypanosoma brucei TBC-B, a key protein involved in the correct chaperoning of quasi-folded tubulin to its native polymerisable state. There is currently little known about this system in trypanosomatids and there were no structures of this protein in protists. Although it was possible to crystallise the full length TbTBC-B protein, no diffraction was obtained. Instead the two domains of the protein were crystallised separately. These are the Ubl and CAP-Gly domains solved to 2.35 and 1.6 Å resolution respectively. It was calculated that the missing linker region most likely contains a small region of a-helical content but is mostly unstructured.The general fold of the Ubl domain is highly conserved, despite low sequence identity between this sequence and other TBC-Bs. Due to this low similarity it was not possible to determine which residues are important for complex formation between this protein and other cofactors or with tubulin.
The CAP-Gly domain despite having higher sequence and structural fold conservation with other TBC-B CAP-Gly domains contains a major difference in the ß7-ß8 tubulin-binding loop. It is postulated this sequence difference of a glycine in the asparagine position of the highly conserved ‘GKNDG’ sequence may affect this domain’s binding to the a-tubulin C-terminal tail. The peptide binding groove of TBC-B forms crystal contact interactions with the C-terminus of a neighbouring molecule. This allowed for the comparison of this binding interaction with the more typical a-tubulin tail binding seen in other CAP-Gly domains. As this protein and domain both elute as monomers from gel filtration columns and this interaction probably represents a crystallographic artefact.
All trypansomatid TBC-B sequences have differences in their tubulin-binding loops and C-terminus that are identical or similar to the a-tubulin tail. In addition to these differences, no other CAP-Gly domains were identified in the trypanosome genome. Although deacetylation is an important modification of tubulin, there are no structures of any tubulin deacetylases and although several histone deacetylase structures are known, none have been reported from protists. Therefore investigations were commenced to identify, clone and obtain recombinant material for crystalisation experiments. A putative tubulin deacetylase form T. brucei was identified and soluble recombinant material could be produced, attempts to crystallise or characterise this protein were prevented by degradation of the protein. At the start of this project, despite tubulin tyrosine ligase (TTL) activity having been identified over thirty years previously, no structures of any TTL enzymes had been determined. It was not possible to produce any material for crystallisation studies from the trypanosomatids therefore other source organisms were investigated. As Sus scrofa TTL had been produced previously in Escherichia coli, it was chosen as a target for crystallisation experiments. However, despite it being possible to obtain folded protein of high purity, no crystals were obtained.
|Date of Award||2012|
|Supervisor||Bill Hunter (Supervisor)|