A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction

Khondker Rufaka Hossain; Daniel R Turkewitz; Stephen A Holt; Leonie Herson; Louise J Brown; Bruce A Cornell; Paul M G Curmi; Stella M Valenzuela

doi:10.1016/j.bbagen.2019.04.020

A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction

Biochim Biophys Acta Gen Subj. 2019 Aug;1863(8):1243-1253. doi: 10.1016/j.bbagen.2019.04.020. Epub 2019 May 8.

Authors

Khondker Rufaka Hossain¹, Daniel R Turkewitz¹, Stephen A Holt², Leonie Herson¹, Louise J Brown³, Bruce A Cornell⁴, Paul M G Curmi⁵, Stella M Valenzuela⁶

Affiliations

¹ School of Life Sciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia.
² Australian Nuclear Science and Technology Organisation (ANSTO), Lucas Height, Australian Centre for Neutron Scattering, New South Wales 2234, Australia.
³ Department of Molecular Sciences, Macquarie University, New South Wales 2109, Australia.
⁴ Surgical Diagnostics Pty Ltd., Roseville, Sydney 2069, Australia; Institute for Biomedical Materials and Devices, University of Technology Sydney, Sydney, New South Wales 2007, Australia.
⁵ School of Physics, The University of New South Wales, Sydney, New South Wales 2052, Australia.
⁶ School of Life Sciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia; Institute for Biomedical Materials and Devices, University of Technology Sydney, Sydney, New South Wales 2007, Australia. Electronic address: stella.valenzuela@uts.edu.au.

PMID: 31075359
DOI: 10.1016/j.bbagen.2019.04.020

Abstract

Background: Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol.

Method: We have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol.

Results: Unlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane.

Conclusion: In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels.

General significance: Members of the CLIC family of proteins demonstrate unusual structural and dual functional properties - as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting activity and a novel regulatory role for cholesterol in such a process.

Keywords: Chloride Intracellular Ion Channel (CLIC) protein; Cholesterol; GXXXG motif; Membrane insertion; Sterol binding motif.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Motifs*
Amino Acid Sequence
Amino Acid Substitution
Cell Membrane / metabolism*
Chloride Channels / chemistry*
Chloride Channels / metabolism
Cholesterol / metabolism*
Conserved Sequence*
Dielectric Spectroscopy
Glycine / chemistry
Humans
Protein Binding
Protein Structure, Secondary

Substances

CLIC1 protein, human
Chloride Channels
Cholesterol
Glycine