The Effect of Mutagenesis and Chaperone Binding on the Insertion Mechanism of Chloride Intracellular Channel 1

Chloride Intracellular Channel 1 (CLIC1) possesses a unique metamorphic capability, enabling it to exist both in a soluble state within the cytosol and to insert into membranes, forming a chloride ion channel, thus exhibiting two native structures. The membrane-bound form is implicated in diseases like Glioblastomas, with inhibition of CLIC1's ion channel activity halting cell proliferation in glioblastoma stem cells. However, the complete membrane insertion mechanism of CLIC1 remains elusive. Oligomerisation is vital for both insertion and chloride conductance, which prompted our mutagenesis studies targeting key CLIC1 regions to elucidate this process. Additionally, we explored potential chaperone binding events involving Heat Shock Protein 90 (HSP90) and Heat Shock Cognate 70 (HSC70) to deepen our understanding of CLIC1's cytosolic function. Utilising biophysical techniques like Nuclear Magnetic Resonance (NMR) and Microscale Thermophoresis (MST), we conducted titration experiments to monitor structural and dynamic changes. Our investigations revealed the interaction of CLIC1 with HSP90, likely binding to CLIC1's N-terminal domain. Furthermore, HSC70 demonstrated nonspecific binding to CLIC1, given the absence of an identified binding site on CLIC1. Despite the nonspecific nature of these interactions, both exhibited strong binding affinities, indicating that these chaperones are potentially involved in CLIC1's insertion mechanism. Mutagenesis experiments demonstrated modulation of CLIC1 dynamics and oligomeric species, identifying the dimerization interface essential for membrane insertion, spanning from Cys24 to Phe66 within the N-terminal domain. These findings offer valuable insights into CLIC1's insertion mechanism and highlight its potential as a therapeutic target in various diseases.