X-ray and neutron reflectivity study of the membrane-bound CLIC1 protein at the air-water interface

Abstract

The CLIC proteins are a ubiquitous family of Chloride Intracellular Ion Channel proteins that are evolutionarily conserved across species and exist as a soluble form and an integral membrane-bound form 1. The X-ray structure of the soluble form of a number of CLIC proteins has been solved and their putative transmembrane domain identified 2-5. However, the factors which facilitate the membrane insertion, the structural transition between these two forms and the structural features of the membrane-bound form for this class of proteins remain largely unknown. In an attempt to answer these questions, we have employed biophysical techniques to study the interaction of wild-type and mutant versions of the protein CLIC1 with monolayers prepared using various mixtures of different phospholipids and sterol molecules. Our findings have demonstrated that cholesterol plays a crucial role for the penetration of CLIC1 into the hydrophobic tails of the lipid monolayer with the protein occupying an area per molecule between 5-7 nm2. We have also demonstrated for the first time that CLIC1 interaction with cholesterol is dependent on the intact 3β-OH group in the sterol ring and that the GXXXG motif in CLIC1 acts as the cholesterol-binding site used by the protein for its initial recognition and binding to membrane cholesterol.