proteins STRUCTURE O FUNCTION O BIOINFORMATICS Comparison of vertebrate and invertebrate CLIC proteins: The crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC Dene R. Littler, 1,2 Stephen J. Harrop, 1,2 Louise J. Brown, 2,3 Greg J. Pankhurst, 2 Andrew V. Mynott, 1,2 Paolo Luciani, 4 Ramya A. Mandyam, 3 Michele Mazzanti, 4 Soichi Tanda, 5 Mark A. Berryman, 6 Samuel N. Breit, 2 and Paul M. G. Curmi 1,2 * 1 School of Physics, University of New South Wales, New South Wales 2052, Australia 2 Centre for Immunology, St Vincent’s Hospital and University of New South Wales, Sydney, New South Wales 2010, Australia 3 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia 4 Department of Molecular Biosciences and Biotechnology, University of Milan, 20133 Milan, Italy 5 Department of Biological Sciences, Molecular and Cellular Biology Program, Ohio University College of Osteopathic Medicine, Athens, Ohio 45701 6 Department of Biomedical Sciences, Molecular and Cellular Biology Program, Ohio University College of Osteopathic Medicine, Athens, Ohio 45701 INTRODUCTION The CLICs are a family of proteins with six human paralogues (CLIC1–6), each of which is conserved in most vertebrate species. The founding family member, p64, was first identified on the basis of its association with chloride ion channel activity and purified from bovine kidney extracts through its affinity to the chloride channel blocker IAA-94. 1–3 Each CLIC protein contains a con- served 230 amino acid C-terminal module (referred to as the ‘‘CLIC module’’) whose structure belongs to the glutathione S- transferase (GST) fold family. 4,5 The functional role(s) of the CLIC proteins is still poorly under- stood. As they were first isolated based on their copurification with an ion channel activity and affinity toward the ion channel blocker IAA-94, one hypothesis is that they may act as, or regulate, chloride channels within intracellular membranes. 3,6–8 Several research groups have demonstrated that CLIC proteins display chloride ion channel activity and in particular, purified recombinant human CLIC1 and CLIC4, expressed in Escherichia coli, produce anion channels in artificial bilayers, in the absence of other proteins. 9–13 Grant sponsors: National Health and Medical Research Council of Australia; Australian Research Council; University of New South Wales; New South Wales Health Research and Development Infrastructure; Access to Major Facilities Program; Italian Ministry of University and Research (MIUR); ‘‘La Sapienza’’ University; Ohio University; Grant sponsor: Wellcome Trust; Grant num- ber: 052458. Dene R. Littler’s current address is The Netherlands Cancer Institute, Division of Molecular Carcinogenesis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. *Correspondence to: Paul M. G. Curmi, School of Physics, University of New South Wales, Sydney, New South Wales 2052, Australia. E-mail: p.curmi@unsw.edu.au Received 23 February 2007; Revised 10 May 2007; Accepted 23 May 2007 Published online 23 October 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21704 ABSTRACT The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Dro- sophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The inverte- brate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the in- vertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cys- teine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing fea- tures of EXC-4 include the fact that it binds to artifi- cial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the sub- stitution of the conserved cysteine by aspartic acid. Proteins 2008; 71:364–378. V VC 2007 Wiley-Liss, Inc. Key words: CLIC; EXC-4; membrane protein; ion channel; GST-fold. 364 PROTEINS V VC 2007 WILEY-LISS, INC.