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A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain

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Abstract

The chloride intracellular channel protein 1 (CLIC1) is unique among eukaryotic ion channels in that it can exist as either a soluble monomer or an integral membrane channel. CLIC1 contains no known membrane-targeting signal sequences and the environmental factors which promote membrane binding of the transmembrane domain (TMD) are poorly understood. Here we report a positively charged motif at the C-terminus of the TMD and show that it enhances membrane partitioning and insertion. A 30-mer TMD peptide was synthesized in which the positively charged motif was replaced by three glutamate residues. The peptide was examined in 2,2,2-trifluoroethanol (TFE), sodium dodecyl sulfate micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using size-exclusion chromatography, far-UV CD, and fluorescence spectroscopy. The motif appears to enhance membrane interaction via electrostatic contacts and functions as an electrostatic plug to anchor the TMD in membranes. In addition, the motif is also involved in orientating the TMD with respect to the cis and trans faces of the membrane. These findings shed light on the intrinsic and environmental factors that promote the spontaneous conversion of CLIC1 from a water-soluble to a membrane-bound protein.

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Abbreviations

CD:

Circular dichroism

CLIC1:

Chloride intracellular channel protein 1

DTNB:

5,5′-Dithio-2-nitrobenzoic acid

DTT:

Dithiothreitol

EEE TMD:

Transmembrane domain of CLIC1 containing Glu49-Glu50-Glu51

λ max :

Emission maximum wavelength

NATA:

N-Acetyl-tryptophanamide

NLS:

Nuclear localization sequence of CLIC4

NRMSD:

Normalized root mean square deviation

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

SDS:

Sodium dodecyl sulfate

TFE:

2,2,2-Trifluoroethanol

TMD:

Transmembrane domain of CLIC1 (residues 24–46)

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Acknowledgments

This work was supported by the University of the Witwatersrand, South African National Research Foundation Grant 68898 to H.W.D and South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation (Grant 64788 to H.W.D). Any opinion, findings, and conclusions or recommendations expressed in this material are those of the author(s) and therefore the National Research Foundation and the Department of Science and Technology do not accept any liability with regard thereto.

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  1. Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2050, South Africa

    Bradley Peter, Sylvia Fanucchi & Heini W. Dirr

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  1. Bradley Peter
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  3. Heini W. Dirr
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Correspondence to Heini W. Dirr.

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Peter, B., Fanucchi, S. & Dirr, H.W. A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain. Eur Biophys J 43, 405–414 (2014). https://doi.org/10.1007/s00249-014-0972-y

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