Inherent flexibility of CLIC6 revealed by crystallographic and solution studies

Abstract

Chloride intracellular channels (CLICs) are a family of unique proteins, that were suggested to adopt both soluble and membrane-associated forms. Moreover, following this unusual metamorphic change, CLICs were shown to incorporate into membranes and mediate ion conduction in vitro, suggesting multimerization upon membrane insertion. Here, we present a 1.8 Å resolution crystal structure of the CLIC domain of mouse CLIC6 (mCLIC6). The structure reveals a monomeric arrangement and shows a high degree of structural conservation with other CLICs. Small-angle X-ray scattering (SAXS) analysis of mCLIC6 demonstrated that the overall solution structure is similar to the crystallographic conformation. Strikingly, further analysis of the SAXS data using ensemble optimization method unveiled additional elongated conformations, elucidating high structural plasticity as an inherent property of the protein. Moreover, structure-guided perturbation of the inter-domain interface by mutagenesis resulted in a population shift towards elongated conformations of mCLIC6. Additionally, we demonstrate that oxidative conditions induce an increase in mCLIC6 hydrophobicity along with mild oligomerization, which was enhanced by the presence of membrane mimetics. Together, these results provide mechanistic insights into the metamorphic nature of mCLIC6.

Conserved cysteine dioxidation enhances membrane interaction of human Cl − intracellular channel 5

“…While hCLIC5-C32S, -C186S, and -C231S exhibited similar response to oxidative conditions as hCLIC5-WT ( Figure S2), the emergence of high-order oligomeric species was completely abolished by the C165S mutation ( Figure 1B). This is in line with lack of oxidationmediated oligomerization of mCLIC6, 8 where this position is naturally substituted with serine (S512; Figure S1), mimicking hCLIC5-C165S. Indeed, mCLIC6-WT demonstrated no oxidation-mediated reactivity ( Figure 1C).…”

“…We have previously shown, using cross-linking, that mCLIC6-WT behaves like hCLIC5-C165S. 8 Similar to the SEC-MALS analysis of its disulfide bridge-mediated oligomerization, introduction of the S512C mutation resulted in a complete recapitulation of both cross-linking and fluorescence profiles observed in hCLIC5-WT ( Figure 2C,F). Specifically, S512C resulted in H 2 O 2 -dependent emergence of high molecular weight bands, which was enhanced in the presence of LUV ( Figure 2C).…”

“…Previously, we suggested that exposure of the inter-domain interface in CLIC6 plays a role in the membrane association process. 8 Importantly, this interface is highly conserved among CLIC family members ( Figure 3B, Figure S1). Here, we studied hCLIC5 to determine whether the involvement of the inter-domain interface can be generalized.…”

“…10 In humans, the CLIC family consists of six isoforms (CLIC1-6). [4][5][6][7][8] While varying in overall lengths, the C-terminal ~220 residues constitute the CLIC homology domain, demonstrating canonical, and highly conserved structural properties. Despite the overwhelming structural similarity between different CLIC isoforms, isoform-specific tissue, and cellular distributions were shown, suggesting a degree of functional specialization is required for various cellular processes involving CLICs.…”

Protein folding and quinary interactions: creating cellular organisation through functional disorder

“…Depending on the conditions (temperature and ionic strength), lymphotactin can adopt one conformation over the other, involving a cooperative global unfolding transition . Several other examples of these metamorphic proteins are reported, in which folding/unfolding events modulate their biological function . In fact, Porter et al .…”