Degree of Conservation between CLICProteins There are currently six knownchloride intracellular ion channel (CLIC) proteins (CLIC1 – CLIC6) which havebeen found in vertebrates, all possessing a high degree of conservation betweenthem, suggesting that they have evolved through duplication from the sameancestral protein in an ancient chordate (Litter et al., 2010). This theory isstrengthened, as urochordate Cionaintestinalis possesses a single CLIC protein which contains a 45% sequenceidentity (highly homologous) to the vertebrate paralogues and also have thecharacteristic three conserved cysteine residues within the sequence. RelatedCLIC proteins have also been found in invertebrates, whose sequences divergeslightly from the vertebrate with a 35% sequence identity and most contain theconserved three characteristic cysteines, with the only exception being somenematodes, where an aspartate takes the place of the active site cysteineresidue (Littler et al., 2010). Function of CLIC Proteins CLIC proteins have only recently beendiscovered and are found to be highly conserved throughout the body butlocalise specifically in various subcellular compartments such as organelles,the cortical actin cytoskeleton, the plasma membrane, vesicles and dependent onthe cell type, centrosomes (Fernandez-Salas et al., 2002; Proutski et al.
,2002; Berryman and Goldenring, 2003; Suh et al., 2003). It has been put in toquestion their proposed function of forming chloride channels, as it isbelieved they can perform alternative cellular processes. Specific biologicalprocesses in which they have been found to be involved in include, keratinocytedifferentiation, apoptosis, receptor trafficking, endothelial vacuole formationand tubulogenesis (Bohman et al., 2005; Suh et al., 2007; Maeda et al., 2008;Tung et al., 2009; Ulmasov et al.
, 2009). CLIC4 has also been linked withmembrane trafficking and / or cytoskeletal trafficking since it is able tointeract with brain dynamin-I in a complex with actin and tubulin (Suginta etal., 2001).
These additional roles of CLIC proteins give more indication whyover or under expression of these proteins is linked to some disease states,including many neurodegenerative disorders and various cancers (Averaimo etal., 2010). Structure of CLIC Proteins CLIC proteins are found under reducing conditions in thecytosol in a soluble globular form, which has a very close structuralsimilarity to the omega-type glutathione S-transferases (GSTs) (Board et al.,2000), and are therefore composed of two domains; an N-terminal thioredoxin-likedomain (residues 16 – 105) that binds to glutathione (GSH) and a highlyconserved all ? helical C-terminal domain, of approximately 240 amino acids,which binds to a second unknown substrate next to GSH to enable them toconjugate.
The second binding site of the xenobiotic compound is less conservedthan the GSH-binding site and is therefore harder to define. The binding siteof GSH closely resembles glutaredoxin and is found highly conserved in allCLICs, suggesting that the chloride ion channel activity is controlled byredox-active signalling molecules in vivo.CLIC1 is able to bind to glutathione covalently in oxidising conditions,through a mixed disulphide bond with the redox active cysteine (Cys24) presentwithin the binding site (Harrop et al., 2001).
Therefore, in a resting cell,the soluble globular form of CLIC1 in the cytosol will be free of glutathioneas the Cys24 will be in a reduced state. In CLIC1 the thiol of Cys24,consisting of the conserved residues Arg29, cis-proline Pro65 and Asp76, isregarded as a highly reactive thiolate possessing a low pKa as a result of itspositioning of helix 1 in the N-terminus and the basic properties of conservedArg29. Hence, it is highly likely that CLIC1 and all other CLIC proteins areGSH-dependent redox-active proteins (Harrop et al., 2001). As seen in figure 1, the structure of CLIC4 shows a break indensity between residues 163 and 173, referring to the flexible foot loop, onlyfound in vertebrate CLIC proteins as GST or invertebrate CLIC proteins do notpossess it. It is located between helix5 and helix 6, with the N-terminus thought to be held in position byinteractions with different residues in close proximity to the reactive Cys35residue of nearby molecules.
It also possesses a joint-like mechanism, as it isable to hinge at residues Pro158 and Arg176 (conserved in all vertebrate CLICproteins (Litter et al., 2005)), whereby the guanidium side chain group ofArg176 enables the generation of a charged hydrogen-bonding network; anidentical structure formation occurs in the CLIC1 protein. CLIC4 also contains a putativeinternal nuclear localisation sequence (NLS) (residues 199 – 206, figure 1), athelix 6 of the C-terminus, whereby its solvent exposed face consists of mainlybasic lysine residues (Lys199, Lys203 and Lys204) with Arg206 positioningitself in the opposing direction to the lysine’s in the loop of helix 6(Littler et al., 2005). The structure of the NLS peptide in CLIC1 is again nearidentical to that of CLIC4 with the only exception being that residue Lys199 isequivalent to Gln188 in CLIC1. The NLS is of high importance to the protein asit is able to control its nuclear import machinery.
This is regulated by helix6 of the NLS partially unfolding to encourage the binding of targetedimportin/karyopherin family proteins to the NLS peptide, to allow furtherconformational changes causing the basic NLS residues to be inserted into theirspecific binding pocket (Litter et al., 2005). CLIC4 is also found in an integralmembrane form and it has been hypothesised that it contains putativetransmembrane (PTM) domain near the N-terminus which runs approximately fromCys35 to Val57 (Figure 1). The presence of a transmembrane region can besupported by performing proteinase K treatment of microsomes containing CLICproteins, resulting in a 27 kDa reduction in size for CLIC4 protein whichleaves only a 6 kDa fragment (Duncan et al.
, 1997). Another notable feature within CLIC proteins isthe presence of several cysteine residues, making them susceptible tointrachain and/or inter-subunit disulphide bond formation, which in some casesaids in the conformational changes required for integral membrane form.