Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 170586, 14 pages http://dx.doi.org/10.1155/2013/170586 Research Article Computational Analysis of the Soluble Form of the Intracellular Chloride Ion Channel Protein CLIC1 Peter M. Jones, 1 Paul M. G. Curmi, 2,3 Stella M. Valenzuela, 1 and Anthony M. George 1 1 School of Medical and Molecular Biosciences, University of Technology Sydney, P.O. Box 123, Broadway, NSW 2007, Australia 2 School of Physics, University of New South Wales, Sydney, NSW 2052, Australia 3 St Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital, Darlinghurst, NSW 2010, Australia Correspondence should be addressed to Anthony M. George; tony.george@uts.edu.au Received 10 April 2013; Revised 26 June 2013; Accepted 27 June 2013 Academic Editor: Serdar Kuyucak Copyright © 2013 Peter M. Jones et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te chloride intracellular channel (CLIC) family of proteins has the remarkable property of maintaining both a soluble form and an integral membrane form acting as an ion channel. Te soluble form is structurally related to the glutathione-S-transferase family, and CLIC can covalently bind glutathione via an active site cysteine. We report approximately 0.6 s of molecular dynamics simulations, encompassing the three possible ligand-bound states of CLIC1, using the structure of GSH-bound human CLIC1. Noncovalently bound GSH was rapidly released from the protein, whereas the covalently ligand-bound protein remained close to the starting structure over 0.25 s of simulation. In the unliganded state, conformational changes in the vicinity of the glutathione- binding site resulted in reduced reactivity of the active site thiol. Elastic network analysis indicated that the changes in the unliganded state are intrinsic to the protein architecture and likely represent functional transitions. Overall, our results are consistent with a model of CLIC function in which covalent binding of glutathione does not occur spontaneously but requires interaction with another protein to stabilise the GSH binding site and/or transfer of the ligand. Te results do not indicate how CLIC1 undergoes a radical conformational change to form a transmembrane chloride channel but further elucidate the mechanism by which CLICs are redox controlled. 1. Introduction Te chloride intracellular channel (CLIC) family of proteins consists of six distinct members in vertebrates: CLIC1-6. Most CLICs are localized to intracellular membranes and are known to participate in protein-protein interactions, particu- larly with cytoskeletal components [1–5]. Te oxidation state of CLIC has been found to afect the activity of associated chloride channels, and CLICs have been shown in vitro to insert from the aqueous phase into phospholipid membranes, where they can function as an anion channel [6, 7]. CLIC proteins can also act as substrates of various kinases [8], and, as has been shown for CLIC5B, tyrosine phosphorylation may afect activity [9, 10]. Although these proteins have been linked to functions including apoptosis and pH and cell cycle regulation [11–13], their exact role in normal cell physiology is uncertain. Te CLIC family is defned by a C-terminal core segment of approximately 230 amino acids that is highly conserved among all family members. Sequences upstream this core vary considerably among the various family members in length and sequence. CLIC1 has been most intensely studied [6, 14–17] and has only a few amino acids upstream of the conserved core that defnes the CLIC family. CLIC1 has been shown to assume both soluble and integral membrane forms, which presumably difer radically in conformation. Te crystal structure of a soluble monomeric form of CLIC1 was found to be a structural homologue of the glutathione-S- Transferase (GST) superfamily of proteins [6]. Tis soluble form of CLIC1 consists of two domains: the N-domain possessing a thioredoxin fold closely resembling glutaredoxin and an all -helical C-domain, which is typical of the GST superfamily (Figure 1). CLIC1 contains an intact glutathione- binding site that was shown to covalently bind glutathione (GSH) via a conserved CLIC cysteine residue, Cys24. Tis led to the suggestion that CLICs are likely to be GSH-dependent redox-active proteins. Te structure of the soluble form of CLIC1 indicates that it closely resembles that of the Omega