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.

Figure 1

The structure of mCLIC6. (a) Cartoon representation of the asymmetric unit from the P 2₁ crystal form showing chain A and B in cyan and green, respectively. (b) Cartoon representation of chain B. The structural motifs of the CLIC domain are labeled. (c) Superposition of the chains A and B from the P 2₁ crystal form. Dashed red lines show distances between the indicated positions.

Figure 2

Surface and inter-domain interface analyses. (a) mCLIC6 electrostatic surface potential. (b) Inter-domain interface hydrophobicity analysis. Hydrophobic patches are shown in red. Positions participating in interface formation are indicated. (c) Detailed illustration of the Q383-T577 hydrogen bond (dashed black line), overlaid with 2Fo – Fc map contoured at 1σ (blue mesh). Indicated residues are shown as sticks and colored by element.

Figure 3

Conservation analysis and disease-associated mutation distribution in mCLIC6. (a) mCLIC6 structure colored according to the conservation score of each residue as determined by Consurf. (b) Conservation score for each residue. Negative values indicate conservation while positive values indicate variability. The sequence refers to the crystallized mCLIC6. (c) Cancer associated mutation positions (green sticks), mapped onto the structure of mCLIC6. A461, involved in both familial goiter and cancer, is shown as orange balls.

Figure 4

Solution properties of mCLIC6. (a) Size exclusion chromatography elution profile of mCLIC6-WT. (b) Experimental SAXS curves of mCLIC6-WT (black line) along with the fit obtained by GNOM (red line) used for ab-initio modelling. (c) Paired-distance distribution function of mCLIC6-WT determined using GNOM. (d) Ab-initio model of CLIC6-WT in solution (grey surface). (e) Size exclusion chromatography elution profile of mCLIC6-Q383A. (f) Experimental SAXS curves of mCLIC6-Q383A (black line) along with the fit obtained by GNOM (red line) used for ab-initio modelling. (c) Paired-distance distribution function of mCLIC6-Q383A determined using GNOM. (d) Ab initio model of CLIC6-Q383A in solution (grey surface). The structure of mCLIC6 was fit into the molecular envelopes using SUPCOMB. The TRX and α domains are presented as blue and red cartoons, respectively.

Figure 5

EOM analysis of mCLIC6. (a) Experimental SAXS curve of mCLIC6 (WT or Q383A; black lines) were fit using CRYSOL or EOM, as indicated. (b) Random Rg pool (black line) and EOM-selected ensemble distributions of mCLIC6-WT (red line) and mCLIC6-Q383A (green line). (c) Random D_max pool (black line) and EOM-selected ensemble distributions of mCLIC6-WT (red line) and mCLIC6-Q383A (green line). (d,e) Representative conformations of mCLIC6-WT (d) and mCLIC6-Q383A (e), found in the crystallographic and EOM analyses. The relative frequency of each conformation in the EOM analysis is indicated along the model Rg in parentheses. The TRX and α domains are presented as blue and red cartoons, respectively.

Figure 6

mCLIC6 response to oxidative environment. (a) The effect of H₂O₂ on mCLIC6, measured using differential scanning fluorimetry in the presence or absence of 5 mM DTT, and presented as Tm difference. (b) Normalized fluorescence-temperature relation of mCLIC6 in the presence of increasing concentrations of H₂O₂. Boltzmann fits used to calculate Tm are colored according to H₂O₂ concentrations. (c) Normalized fluorescence-temperature relation of mCLIC6-Q383A in the presence of increasing concentrations of H₂O₂. (d) Stern–Volmer plots for mCLIC6 tryptophan fluorescence quenching by acrylamide in both the absence and presence of H₂O₂.

Figure 7

mCLIC6 oligomerization and membrane association. (a) Cysteine exposure (green), demonstrated using surface representation of mCLIC6 structure (grey). Note the C533 is inaccessible to the external solution. (b) Analytical size exclusion chromatography in the presence of increasing H₂O₂ concentrations. (c) SDS-PAGE of mCLIC6, following DSS-mediated cross-linking in the presence of H₂O₂ and LUVs. Red circles represent visible oligomeric states of the protein (n = 3). (d) mCLIC6 tryptophan fluorescence decay kinetics in the absence or presence of H₂O₂ and LUVs.