Structure of the Janus Protein Human CLIC2

doi:10.1016/j.jmb.2007.09.041

Journal of Molecular Biology

Volume 374, Issue 3, 30 November 2007, Pages 719-731

https://doi.org/10.1016/j.jmb.2007.09.041 Get rights and content

Abstract

Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-Å resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions. We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction.

Section snippets

Overall Structure of Human CLIC2

We determined the structure of human CLIC2 from two crystal forms, denoted A and B, to a resolution of 1.8 Å (Table 1). The CLIC2 molecule is box shaped (∼60 × 60 × 35 Å), consisting of two domains (Fig. 1a and b). The N-terminal domain (residues 1–94) adopts a thioredoxin-like fold consisting of a four-stranded mixed β-sheet with two α-helices running parallel with the sheet on one face (α1 and α3) and one helix (α2) running perpendicular to the sheet on the other face. The electron density for

Similarity to Other CLIC Structures

The structures of two other CLIC proteins have been determined: human CLIC17., 11. and human CLIC4.31., 34. CLIC2 shares 60% sequence identity with CLIC1 and 63% sequence identity with CLIC4. The overall structures are similar, with an overall rmsd between CLIC2 and CLIC1 [Protein Data Bank (PDB) code: 1K0M] on α-carbon atoms of 1.8 Å (for 224 matching residues) or 0.9 Å if the foot loop region is excluded (Fig. 1e) and an overall rmsd between CLIC2 and CLIC4 (PDB code: 2AHE) on α-carbon atoms

Similarity to GSTs

CLIC2 adopts the same topology of secondary structure as GSTs and most closely resembles beta-, omega-, tau-, and zeta-GSTs with an rmsd on α-carbon atoms of approximately 2 Å against each GST. This value is well within the range found for the superposition of GSTs from different classes.²⁸ Four key residues are conserved in virtually all GSTs and CLICs: a cis-proline residue (Pro71 in CLIC2) that provides the correct active site geometry for binding GSH in GSTs, an aspartic acid residue

Influence of the Intramolecular Disulfide on Oligomer Formation

The structure of CLIC2 revealed an intramolecular disulfide bridge between Cys30 and Cys33 (Fig. 1d). In contrast, CLIC1 was shown to have no disulfide in the monomer state, but an intramolecular disulfide formed in the dimer state between residues Cys24 and Cys59.¹¹ The corresponding residues in CLIC2 are Cys30 and Ala65, and hence such a disulfide is not possible in CLIC2. Nevertheless, we wanted to examine whether CLIC2 could undergo the same dramatic changes in conformation by engineering

Chloride Efflux Experiments

Studies have shown that CLIC1 and CLIC4 produce chloride efflux from artificial liposomes, with the flux increasing at acidic pH levels.10., 11., 31. The flux data for human CLIC2 appear to be very similar to what were observed for CLIC1 and CLIC4,¹¹^,³¹ except with a small notable pH shift in its response (Fig. 3a). The CLIC2 flux is minimal at pH 8.0 and increases markedly with decreasing pH levels. The other CLIC proteins differ in the position of the efflux minimum, which occurs between pH

Channel Activity

Single-channel currents were observed when soluble CLIC2 was added to artificial lipid bilayers using tip–dip electrophysiology (Fig. 3b). The single-channel conductance was measured at 48.4 ± 1.2 pS, which is higher than the conductance measurements observed for CLIC1 (28 ± 9 pS)11., 42. and CLIC4 (30 ± 2 pS)³¹ under similar conditions (Fig. 3c). The probability of observing single-channel currents after the addition of CLIC2 to the tip–dip chamber shows a strong pH dependence, with no current

Functional Consequences of Similarities to Other Proteins

A search of the PDB for similar folds to CLIC2 showed that it most closely resembles the beta-, omega-, tau-, and theta-GSTs (after omitting hits to other CLIC proteins). Despite the structural similarities between GSTs and CLICs, CLIC2 has been shown to display a low affinity for GSH and is not retained on GSH affinity columns.³² Neither did we see GSH bound to the mouth region in either crystal form despite its being an essential ingredient in the crystallization of both forms. Furthermore,

CLIC2 Recognition of Binding Partners

The insertion of the foot loop into the CLIC2 mouth region of neighboring molecules, as observed for CLIC4,³¹ suggests that CLIC2 might bind other proteins. By analogy with thioredoxins, Harrop et al.⁷ postulated that the wide mouth region of CLICs might accommodate a putative protein target. Within the CLIC family, there is a relatively low sequence conservation of the foot loop, although it does tend to possess a net negative charge. The conformations of the foot loop regions that enter the

Concluding Remarks

In this present study, we determined the crystal structure of human CLIC2. We showed that it adopts a very similar fold to the CLIC1 and CLIC4 proteins, with the major differences being in the mobile helix α2 region and the foot loops. Two unusual features of the CLIC2 structure are the presence of an intramolecular disulfide in the N-terminal domain and the insertion of the foot loop regions into the mouth regions of neighboring molecules in the crystal lattice. We demonstrated for the first

Materials and Methods

Details of the cloning, protein expression, and purification have been described previously.³² Briefly, CLIC2 was expressed in E. coli BL21(DE3) cells as an N-terminal His-tagged fusion protein and purified by Ni affinity chromatography. The protein was further purified by gel filtration and then dialyzed into 50 mM of Hepes, pH 7.5, and 100 mM of NaCl and concentrated to 7.3 mg/ml for crystal form A and into 20 mM of Tris–HCl, pH 7.5, and 50 mM of NaCl and concentrated to 15 mg/ml for crystal

Protein Data Bank accession numbers

The atomic coordinates and structure factors (accession codes 2R4V and 2R5G for crystal forms A and B, respectively) have been deposited in the PDB of the Rutgers University Research Collaboratory for Structural Bioinformatics (New Brunswick, NJ, USA)‡.

Acknowledgements

This work, including use of the BioCARS sector, was supported by the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia under the Major National Research Facilities Program. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research. This work was also supported by grants from the National Health and Medical Research Council of Australia (NHMRC) and Australian Research Council (ARC) to

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^†: Joint first co-author.

²: Present addresses: B. A. Cromer, Howard Florey Institute, University of Melbourne, Victoria 3010, Australia.

³: J.J. Adams, Australian Synchrotron Project, 800 Blackburn Road, Clayton, Victoria 3168, Australia.

⁴: D. R. Littler, Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

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