The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition.

Dene R Littler,W Douglas Fairlie,Raffaella Tonini,Asne R Bauskin,Samuel N Breit,Terence J Campbell,Louise J Brown,Paul M.G Curmi,Matthew Z Demaere,Stephen J Harrop,Susan Pankhurst,Greg J Pankhurst,Michele Mazzanti

doi:10.1074/jbc.m308444200

Abstract

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.

Highlights

Most proteins adopt a well defined three-dimensional structure; it is increasingly recognized that some proteins can exist with at least two stable conformations
Reversible Redox-induced Dimerization of CLIC1—A feature of CLIC1 is that it possesses a glutaredoxin-like active site, suggesting that its function may be under redox control [8]
To investigate this we have studied the effect of reactive oxygen species on CLIC1

Summary

EXPERIMENTAL PROCEDURES

Expression, Purification, and Oxidation of CLIC1—Recombinant GST-CLIC1 fusion proteins (wild type and mutants) were expressed in. Protein was further purified at 4 °C by gel filtration on a Superdex G75 column (Amersham Biosciences) preequilibrated with 10 mM Hepes, 100 mM KCl, 0.5 mM CaCl2, 1 mM DTT, and 1 mM NaN3 at pH 7.0. The resulting peak at 30 kDa corresponding to monomeric CLIC1 was dialyzed at 4 °C into 1ϫ phosphatebuffered saline and concentrated to 15 mg/ml. For gel filtration chromatography analysis untreated and oxidized proteins were diluted to 200 ␮l final volume before injection onto a Superose 12 (HR 10/30, Amersham Biosciences) column preequilibrated in phosphate-buffered saline, pH 7.4, at 0.5 ml/min. The dimeric fraction was passed over the Superdex G75 column a second time, and the resulting single 60kDa peak was concentrated, flash-frozen in liquid nitrogen, and stored at Ϫ80 °C. R factor (Rfree) r.m.s.d. bond lengthsa r.m.s.d. bond anglesa Ramachandran plota

Additionally allowed

Bilayer electrophysiologyb

RESULTS

DISCUSSION