Transmembrane Extension and Oligomerization of the CLIC1 Chloride Intracellular Channel Protein upon Membrane Interaction Sophia C. Goodchild, † Christopher N. Angstmann, ‡ Samuel N. Breit, § Paul M. G. Curmi, §,⊥ and Louise J. Brown* ,† † Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia ‡ School of Mathematics and Statistics, University of New South Wales, New South Wales 2052, Australia § St. Vincent’s Centre for Applied Medical Research, St. Vincent’s Hospital, Sydney, NSW 2010, Australia ⊥ School of Physics, University of New South Wales, New South Wales 2052, Australia * S Supporting Information ABSTRACT: Chloride intracellular channel proteins (CLICs) differ from most ion channels as they can exist in both soluble and integral membrane forms. The CLICs are expressed as soluble proteins but can reversibly autoinsert into the membrane to form active ion channels. For CLIC1, the interaction with the lipid bilayer is enhanced under oxidative conditions. At present, little evidence is available characterizing the structure of the putative oligomeric CLIC integral membrane form. Previously, fluorescence resonance energy transfer (FRET) was used to monitor and model the conformational transition within CLIC1 as it interacts with the membrane bilayer. These results revealed a large-scale unfolding between the C- and N-domains of CLIC1 as it interacts with the membrane. In the present study, FRET was used to probe lipid-induced structural changes arising in the vicinity of the putative transmembrane region of CLIC1 (residues 24-46) under oxidative conditions. Intramolecular FRET distances are consistent with the model in which the N-terminal domain inserts into the bilayer as an extended α-helix. Further, intermolecular FRET was performed between fluorescently labeled CLIC1 monomers within membranes. The intermolecular FRET shows that CLIC1 forms oligomers upon oxidation in the presence of the membranes. Fitting the data to symmetric oligomer models of the CLIC1 transmembrane form indicates that the structure is large and most consistent with a model comprising approximately six to eight subunits. T he chloride intracellular channels (CLICs) are a class of ubiquitously expressed anion channels possessing both enigmatic structural and functional properties. CLICs differ from classical ion channels as they are expressed as soluble proteins and lack both a leader sequence for membrane targeting and the multiple hydrophobic transmembrane domains, typical of classical ion channel proteins. 1 The CLIC proteins can undergo autonomous membrane insertion to form integral membrane ion channels. 2 Because of their ability to reversibly interconvert between multiple functional structures using the same amino acid sequence, members of the CLIC family have recently been described as part of the novel metamorphic protein class. 3,4 The restricted divergence and high level of conservation shared among CLIC proteins attests to a significant cellular function. However, despite increasing data describing their physiological function 5-9 and disease associations including cancer 10-12 and more recently intellectual disability, 13 their molecular function remains unclear. In addition to ion channel activity, many other functional roles have been ascribed to the CLIC proteins. These include redox regulation, enzymatic, and transcription factor activity. Several CLIC members have also been described as scaffolding proteins thought to couple the membrane to the cytoskeleton (for further detailed review, see ref 2). However, the precise role of both the soluble and integral membrane forms of CLIC1 is not fully understood. A complete understanding of the biological importance of the metamorphic transitions that occur as CLIC1 inserts into the bilayer is also hampered by a lack of structural evidence supporting a multimeric integral membrane state as would be necessary for ion channel conductance. To date, the crystal structures of four soluble monomeric vertebrate CLICsCLIC1 (Figure 1a), 14 CLIC2, 15 CLIC3, 16 and CLIC4 17 and two invertebrate CLIC-like proteins Drosophila melanogaster DmCLIC and Caenorhabditis elegans EXC4 18 have been determined. These soluble, monomeric CLIC proteins have all been found to adopt a conserved glutathione S-transferase (GST) family fold consisting of an N- terminal thioredoxin domain and a compact, all-helical, C- terminal domain. Received: August 10, 2011 Revised: November 4, 2011 Article pubs.acs.org/biochemistry © XXXX American Chemical Society A dx.doi.org/10.1021/bi2012564 | Biochemistry XXXX, XXX, XXX-XXX