X-ray crystallography helps reveal role of ‘CLIC’ proteins

Chloride Intracellular Channels (CLIC) are an intriguing family of proteins initially discovered in the 1980s during research linked to cystic fibrosis [1]. While originally thought to function as chloride channels, subsequent research has revealed that CLIC proteins can share a globular form and exhibit diverse functions not related to chloride ion transport [2].

CLICs have been implicated in various cellular processes, including ion channel regulation, membrane trafficking and cell cycle regulation. They also demonstrate a unique capacity to transition between soluble and membrane-associated states, suggesting roles in membrane remodelling and cell signalling. Despite extensive study, precise understanding of the physiological functions of CLIC proteins remained incomplete.

Investigating the role of CLICs

Aiming to uncover the elusive physiological role of CLICs, a team of researchers from Israel and the Czech Republic employed a comprehensive array of biochemical, biophysical and structural techniques, including X-ray crystallography at beamline ID30B, focusing on the human CLIC5 as their case study. They revealed direct interactions between CLICs and the membrane, and their previously unrecognized capacity to facilitate cell membrane fusion, both in vitro and in vivo (Figure 1a). 

Fig. 1: Model and in vitro analysis of CLIC-induced membrane fusion. a) CLICs are schematically represented as circles comprising the TRX (blue) and α (red) domains. A triggering event results in exposure of the inter-domain interface, favouring membrane interaction and, subsequently, fusion. b) Spectral FRET-based analysis of CLIC5 membrane interaction. Averaged fluorescence-emission spectra were collected using excitation at F₂₈₀ at the indicated incubation times, where CLIC5 tryptophan residues serve as energy donors (the peak at F₃₄₄), and dansyl-PE-containing liposomes are the FRET acceptors (F₅₀₅, dansyl-PE emission due to the occurrence of FRET; inset). c) DLS analysis revealed that a population with a significantly increased radius emerged only in the presence of CLIC5.

First, purified CLIC5 was added to membrane liposome-containing solution, giving rise to an unexpected observation – the opacity of the solution markedly increased upon the addition of CLIC5, indicating its potential impact on liposome size. Next, the direct interaction between CLICs and membrane liposomes was confirmed by biochemical assays that showed CLIC5’s strong affinity for lipid bilayers, while fluorescence-resonance energy transfer (FRET) between CLIC5 and liposomes (Figure 1b) provided further evidence. Additionally, dynamic light scattering (DLS) analysis revealed a significant increase in liposome size when exposed to CLIC5 (Figure 1c), indicating potential involvement in processes such as fusion. Further fluorescent-based assays confirmed CLIC5’s ability to induce complete fusion of nearby membranes. This novel ‘fusogenic’ property was enhanced under acidic pH conditions, a known trigger for membrane translocation of CLIC proteins that is common across the CLIC protein family. 

How do CLIC proteins promote fusion?

Next, to understand how CLIC proteins facilitate membrane fusion, the researchers identified a mutant called F34D that abolishes this activity (Figure 2a,b). They used X-ray crystallography at the beamline ID30B, alongside hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses, to determine the structural elements crucial for membrane fusion and response to environmental cues (Figure 2a). Although the crystal structure of the F34D mutant closely resembled that of wild-type CLIC5, it revealed the formation of an inter-domain salt bridge, supported by molecular dynamics simulations. Complementary HDX-MS analysis revealed conformational changes upon exposure to acidic pH, which were largely prevented by the F34D mutation.

Fig. 2: The inter-domain interface is crucial for CLIC-mediated membrane fusion. a) Crystal structure of CLIC5-Δloop-F34D, obtained using ESRF ID30B (left). 2Fo-Fc electron density map, contoured at 1σ, is provided for position 34, and surrounding residues within 5 Å are shown as sticks (right). b) Averaged fluorescence-emission spectra following excitation at F₂₈₀ for CLIC5-F34D (red) and CLIC5-WT (black; as in Figure 1b) after incubation of 75 min. CLIC5-F34D exhibits a markedly reduced FRET with dansyl-PE-containing liposomes (left) and liposomal diameter, as assessed by DLS (right). c) Excretory canal defects in the nematode C. elegans with point mutation F38D in the exc-4 gene. Worm cartoon (top) and representative images of L4 stage worms expressing an excretory canal fluorescent marker (green) (bottom). Control worm (exc-4 WT) with an excretory canal extending from the pharynx to the tail and exc-4 mutant worm (exc-4 F38D) with an excretory canal extending only to the vulva and harbouring visible cysts along the pharyngeal region are shown. Yellow arrows indicate the end of the canal in each worm.

This suggests that the exposure of the inter-domain interface is a critical conformational change required for membrane interaction and subsequent fusion. Remarkably, when the researchers applied the same mutation to the CLIC orthologue in the in vivo C. elegans model using CRISPR, they observed impaired formation of the excretory canal, a process known to require extensive membrane remodelling (Figure 2c). These results provide further evidence of the importance of CLICs’ fusogenic activity in various physiological processes. 

In conclusion, this study proposes a mechanistic model that explains CLICs’ role as fusogens (Figure 1a). Initially, most CLIC proteins have a globular shape, which hides the hydrophobic inter-domain interface and prevents interaction with membranes. However, when exposed to stimuli such as acidic pH or reactive oxygen species [3], they undergo a transformation to an elongated form, revealing the hydrophobic interface enabling membrane association. As a result, CLIC subunits on the membrane assemble into oligomeric complexes, which facilitate the approximation and destabilization of adjacent membranes, overcoming the energy barrier required for complete fusion. This model sheds light on the physiological functions of CLICs and serves as a basis for further exploration of their involvement in mediating membrane fusion events across various cellular processes.

 
Principal publication and authors
Chloride intracellular channel (CLIC) proteins function as fusogens, B. Manori (a), A. Vaknin (b), P. Vaňková (c), A. Nitzan (d), R. Zaidel-Bar (d), P. Man (e), M. Giladi (a,f), Y. Haitin (a,g), Nat. Commun. 7, 15(1), 2085 (2024); https://doi.org/10.1038/s41467-024-46301-z
(a) Department of Physiology and Pharmacology, Faculty of Medicine, Tel Aviv University (Israel)
(b) School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University (Israel)
(c) Institute of Biotechnology of the Czech Academy of Sciences, BioCeV, Vestec (Czech Republic)
(d) Department of Cell and Developmental Biology, Faculty of Medicine, Tel Aviv University (Israel)
(e) Institute of Microbiology of the Czech Academy of Sciences, Division BioCeV, Vestec (Czech Republic)
(f) Tel Aviv Sourasky Medical Center, Tel Aviv (Israel)
(g) Sagol School of Neuroscience, Tel Aviv University (Israel)

References
[1] D.W. Landry et al., Science 244, 4911, 1469-1472 (1989).
[2] E. Argenzio & W.H. Moolenaar, J. Cell Sci. 129, 22, 4165-4174 (2016).
[3] A. Ferofontov et al., FASEB J., 34, 8, 9925-9940 (2020).

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