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

Abstract

The human chloride intracellular channel (hCLIC) family is thought to transition between globular and membrane-associated forms by exposure of a hydrophobic surface. However, the molecular identity of this surface, and the triggering events leading to its exposure, remain elusive. Here, by combining biochemical and structural approaches, together with mass spectrometry (MS) analyses, we show that hCLIC5 is inherently flexible. X-ray crystallography revealed the existence of a globular conformation, while small-angle X-ray scattering showed additional elongated forms consisting of exposure of the conserved hydrophobic inter-domain interface to the bulk phase. Tryptophan fluorescence measurements demonstrated that the transition to the membrane-associated form is enhanced by the presence of oxidative environment and lipids. Using MS, we identified a dose-dependent oxidation of a highly conserved cysteine residue, known to play a key role in the structurally related omega-class of glutathione-S-transferases. Hydrogen/deuterium exchange MS analysis revealed that oxidation of this cysteine facilitates the exposure of the conserved hydrophobic inter-domain interface. Together, our results pinpoint an oxidation of a specific cysteine residue as a triggering mechanism initializing the molecular commitment for membrane interaction in the CLIC family.

TTYH family members form tetrameric complexes at the cell membrane

“…These files were then loaded to DeutEx software 49 together with the sequence of the mTTYH1 construct and a list of identified peptides (see below). The processed HDX data were visualized in PyMol and using MSTools ( http://peterslab.org/MSTools/index.php ) 50 , and a back-exchange correction was done 51 , 52 . Peptide identification for mTTYH1 dimeric and tetrameric populations was done using the same LC setup as described above but with the ESI-TIMS ToF Pro PASEF (Bruker Daltonics).…”

Inflammasomes as regulators of mechano-immunity

“…The role of chloride efflux downstream of classical NLRP3 activators has been reported in other studies (Domingo-Fernandez et al, 2017 ; Green et al, 2018 ; Mayes-Hopfinger et al, 2021 ; Tang et al, 2017 ), and likely involves other chloride channels, such as chloride intracellular channels (CLICs). CLICs make up a family of six members (Ferofontov et al, 2020 ; Landry et al, 1989 ). They are intracellular globular proteins, that undergo extensive conformational change in response to changes in oxidative and acidic environments inside the cell.…”

“…They are intracellular globular proteins, that undergo extensive conformational change in response to changes in oxidative and acidic environments inside the cell. The conformational change exposes a hydrophobic patch in CLIC proteins, which allows their membrane insertion, oligomerisation, and channel formation (Al Khamici et al, 2016 ; Ferofontov et al, 2018 ; Ferofontov et al, 2020 ; Goodchild et al, 2011 ; Goodchild et al, 2009 ; Nesiu et al, 2019 ). Once in the membrane of activated macrophages CLICs amplify TLR4 signalling, inflammasome activation and IL-1β secretion (Domingo-Fernandez et al, 2017 ; Green et al, 2018 ; Tang et al, 2017 ).…”

Chloride intracellular channel (CLIC) proteins function as fusogens

“…The exposure of CLICs to membrane mimetics was previously shown to result in their conformational alteration and oligomerization 11,13,35,36 . However, the possible reciprocal effects conferred by CLICs on the membrane were not investigated.…”

“…Moreover, subsequent structural investigations using X-ray crystallography revealed a globular conformation of the soluble form, composed of an N-terminal thioredoxin domain (TRX) followed by a C-terminal α domain [7][8][9][10][11][12][13] .…”