Sterol Structural Features' Impact on the Spontaneous Membrane Insertion of CLIC1 into Artificial Lipid Membranes

Background: A membrane protein interaction with lipids shows distinct specificity in terms of the sterol structure. The structure of the sterol's polar headgroup, steroidal rings, and aliphatic side chains have all been shown to influence protein membrane interactions, including the initial binding and subsequent oligomerization to form functional channels. Previous studies have provided some insights into the regulatory role that cholesterol plays in the spontaneous membrane insertion of the chloride intracellular ion channel protein, CLIC1. However, the manner in which cholesterol interacts with CLIC1 is yet largely unknown. Method: In this study, the CLIC1 interaction with different lipid:sterol monolayers was studied using the Langmuir trough and neutron reflectometry in order to investigate the structural features of cholesterol essential for the spontaneous membrane insertion of the CLIC1 protein. Molecular docking simulations were also performed to study the binding affinities between CLIC1 and the different sterol molecules. Results: This study, for the first time, highlights the vital role of the free sterol 3β-OH group as an essential structural requirement for the interaction of CLIC1 with cholesterol. Furthermore, the presence of additional hydroxyl groups, methylation of the sterol skeleton, and the structure of the sterol alkyl side chain have also been shown to modulate the magnitude of CLIC1 interaction with sterols and hence their spontaneous membrane insertion. This study also reports the ability of CLIC1 to interact with other naturally existing sterol molecules. General Significance: Like the sterol molecules, CLIC proteins are evolutionarily conserved with almost all vertebrates expressing six CLIC proteins (CLIC1-6), and CLIC-like proteins are also present in invertebrates and have also been reported in plants. This discovery of CLIC1 protein interaction with other natural sterols and the sterol structural requirements for CLIC membrane insertion provide key information to explore the feasibility of exploiting these properties for therapeutic and prophylactic purposes.