Membrane mimetics induce helix formation and oligomerization of the chloride intracellular channel protein 1 transmembrane domain.

ABSTRACT

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The transmembrane domain (TMD), implicated in membrane penetration and pore formation, comprises helix α1 and strand ß2 of the N-domain of soluble CLIC1. The mechanism by which the TMD binds, inserts, and oligomerizes in membranes to form a functional chloride channel is unknown. Here we report the secondary, tertiary, and quaternary structural changes of the CLIC1 TMD as it partitions between an aqueous and membrane-mimicking environment. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate (SDS) micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes using far-ultraviolet circular dichroism and fluorescence spectroscopy. Data obtained in the presence of SDS micelles and POPC liposomes show that Trp35 and Cys24 have reduced solvent accessibility, indicating that the peptide adopts an inserted orientation. The peptide assumes a helical structure in the presence of these mimetics, consistent with its predicted membrane conformation. This acquisition of secondary structure is concentration-dependent, suggesting an oligomerization event. Stable dimeric and trimeric species were subsequently identified using SDS-polyacrylamide gel electrophoresis. We propose that, in the vicinity of membranes, the mixed α/ß TMD in CLIC1 rearranges to form a helix that then likely dimerizes via noncovalent helix-helix interactions to form a membrane-competent protopore complex. Such oligomerization would be essential for forming a functional ion channel, given that each CLIC1 monomer possesses only a single TMD. This work highlights the central role of the TMD in CLIC1 function It is capable of promoting membrane insertion and dimerization in the absence of the C-domain and large portions of the N-domain.