pubs.acs.org/Biochemistry Published on Web 05/27/2010 r 2010 American Chemical Society 5278 Biochemistry 2010, 49, 5278–5289 DOI: 10.1021/bi100111c Metamorphic Response of the CLIC1 Chloride Intracellular Ion Channel Protein upon Membrane Interaction † Sophia C. Goodchild, ‡ Michael W. Howell, ‡ Dene R. Littler, §,# Ramya A. Mandyam, ‡,r Kenneth L. Sale, ) Michele Mazzanti, ^ 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 Physics, University of New South Wales, Sydney, New South Wales 2052, Australia, ) Biosystems R&D Department, Sandia National Laboratories, Livermore, California 94551, ^ Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy, and @ St. Vincent’s Centre for Applied Medical Research, St. Vincent’s Hospital, and University of New South Wales, Sydney, New South Wales 2010, Australia # Current address: The Netherlands Cancer Institute, Division of Molecular Carcinogenesis, Plesmaanlaan 121, 1066 CX Amsterdam, The Netherlands. r Current address: Institute for Biomolecular Sciences, Division of Molecular Cell Biology, QBP, University of Queensland, Brisbane, QLD 4072, Australia. Received January 25, 2010; Revised Manuscript Received April 29, 2010 ABSTRACT: A striking feature of the CLIC (chloride intracellular channel) protein family is the ability of its members to convert between a soluble state and an integral membrane channel form. Direct evidence of the structural transition required for the CLIC protein to autonomously insert into the membrane is lacking, largely because of the challenge of probing the conformation of the membrane-bound protein. However, insights into the CLIC transmembrane form can be gained by biophysical methods such as fluorescence resonance energy transfer (FRET) spectroscopy. This approach was used to measure distances from tryptophan 35, located within the CLIC1 putative N-domain transmembrane region, to three native cysteine residues within the C-terminal domain. These distances were computed both in aqueous solution and upon the addition of membrane vesicles. The FRET distances were used as constraints for modeling of a structure for the CLIC1 integral membrane form. The data are suggestive of a large conformational unfolding occurring between the N- and C-domains of CLIC1 upon interaction with the membrane. Consistent with previous findings, the N-terminal domain of CLIC1 is likely to insert into the lipid bilayer, while the C-domain remains in solution on the extravesicular side of the membrane. Traditionally, it has been thought that proteins adopt a well- defined tertiary structure, with at most small conformational changes accompanying function. However, it is becoming increas- ingly apparent that some proteins can shift between two or more stable conformations, a property termed “metamorphic” (1). This metamorphic property is a prominent feature of the unique CLIC 1 (chloride intracellular channel) protein family. In stark contrast to members of the traditional ion channel families, the CLICs are expressed as soluble proteins, with a conserved glutathione S-transferase (GST) superfamily-like fold that lacks both a leader sequence for membrane targeting and a clearly defined hydro- phobic transmembrane domain. However, integration of recom- binant CLIC protein into artificial, synthetic lipid bilayers to display ion channel activity has been demonstrated for CLIC members (2-10). In addition, recent evidence has suggested other canonical GST proteins may also possess membrane-transversing properties (11). However, both the biochemical and structural bases of the novel, automomous mechanism of CLIC membrane insertion remain unclear. Since identification of the first CLIC protein and its mRNA transcript in humans in 1997 (12), much has been learned about this highly conserved six-member protein family (CLIC1-6). CLIC homologues have also been identified in invertebrates such as nematodes (13) and insects (14) and, more recently, in plants (15). Each CLIC homologue possesses a highly conserved C-terminal “CLIC module” of ∼240 amino acids, with some members also possessing additional, unrelated N-terminal domains. Overall, the high degree of sequence similarity shared among members of the CLIC family and preservation of a defined set of CLIC orthologues throughout vertebrate evolution intuitively implies evidence of their physiological importance (16). Although a clear elucidation of the molecular, cellular, and physiological function of the CLIC proteins, including their ion conducting role, is still in its infancy, members of the CLIC family have also been implicated in many fundamental processes, including cellular division (17, 18), apop- tosis and response to DNA damage (19-21), bone resorption (22), and tubulogenesis (13, 23-26), to name a few. Because of the difficulty in obtaining direct structural data of the transmembrane CLIC state, structural characterization of the CLICs has so far been limited to the soluble form. The soluble monomeric GST-like CLIC fold consists of two domains: a † Supported by an Australian Research Council (ARC) grant and an ARC APD fellowship to L.J.B. *To whom correspondence should be addressed: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. Telephone: þ61 2 9850 8294. Fax: þ61 2 9850 8313. E-mail: Louise.Brown@mq.edu.au. 1 Abbreviations: CLIC, chloride intracellular channel; FRET, fluores- cence resonance energy transfer; EPR, electron paramagnetic resonance; PTM, putative transmembrane region encompassing Cys24-Val46 in CLIC1; WT, wild type; IAEDANS, 5-(iodoacetamidoethyl)amino- naphthalene-1-sulfonic acid; BDMC, 4-bromomethyl-6,7-dimethoxycou- marin; HNB, 2-hydroxy-5-nitrobenzene; MTSSL, 3-methylthiosulfonyl- 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline.