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Abstract
The chloride intracellular channel (CLIC) family of proteins are an unusual class of chloride channels that possess the ability to auto-insert into cellular membranes. The CLICs exhibit ubiquitous tissue and cellular distributions and adopt a glutathione S-transferase fold in the soluble form that is highly conserved in vertebrates. CLIC homologues have been identified in the model organisms C. elegans and D. melanogaster, and in the former case have been extensively characterised in regards to function. In this thesis, we present the crystal structure of the D. melanogaster CLIC, revealing several unique features in the conserved invertebrate CLIC fold including an elongated C-terminal extension and metal binding site. The bound metal is identified as the potassium cation, resolving concerns regarding previously published work that assign the metal as the isoelectronic calcium cation.
It has been reported that a human CLIC protein, CLIC4, translocates to the nucleus in response to cellular stress, facilitated by a putative CLIC4 nuclear localisation signal (NLS). The CLIC4 NLS adopts α-helical structure in the native CLIC4 structural fold. It is proposed that CLIC4 is transported to the nucleus via the classical nuclear import pathway after binding the import receptor, importin-α. We have determined the X-ray crystal structure of a truncated form of importin-α bound to a CLIC4 NLS bearing peptide. The NLS peptide binds the major binding site in an extended conformation similar to that observed for the classical SV40 large T-antigen NLS. A tyrosine residue within the CLIC4 NLS makes surprisingly favourable interactions by forming side chain hydrogen bonds to the importin-α backbone. This structural evidence supports the hypothesis that CLIC4 translocation to the nucleus is governed by the importin-α nuclear import pathway, providing it can undergo a conformational rearrangement that exposes the NLS in an extended conformation.
We further analyse importin-α:NLS binding interactions by solving high resolution structures of truncated importin-α containing an empty binding site and bound to the SV40 NLS. A surprising interaction is discovered between importin-α and an NLS-like motif in the endogenous E. coli 30S ribosomal subunit S21, revealing new insight into importin-α recognition of full length cargo.