Mechanistic Elucidation of CLIC1 Membrane Insertion via Structural and Dynamic Modulation

Chloride Intracellular Channel 1 (CLIC1) is a metamorphic protein capable of transitioning from a soluble cytoplasmic state to a membrane-bound chloride channel. This conformational shift, crucial for physiological processes such as cell volume regulation, electrical excitability, and angiogenesis, is linked to pathological conditions including malignancies and cardiovascular diseases. Despite its significance, the molecular mechanism driving CLIC1s membrane insertion has remained elusive. Using an integrated structural biology approach combining NMR spectroscopy, SAXS, biophysical methods, and mutagenesis, we uncover the dynamic landscape underpinning CLIC1 function. Solution NMR and SAXS reveal that CLIC1 adopts a conformational ensemble in equilibrium, characterized by a compact ground state and a partially extended state exposing key membrane-interacting regions. Zn2+ binding acts as a critical trigger, inducing structural rearrangements, increasing protein flexibility, and promoting oligomerization essential for membrane insertion. Our findings demonstrate that structural flexibility, particularly within dynamic loop regions and interdomain linkers, is intrinsic to CLIC1s ability to adapt to membrane interactions. Zn2+-induced dimerization and tetramerization were identified as key steps preceding insertion, with mutations in the transmembrane (TM) region revealing pivotal roles for residues R29 and W35 in modulating protein dynamics, oligomerization and insertion eXiciency. This study provides a mechanistic framework for CLIC1s transition to its membrane-bound state, oXering insights into the interplay between conformational dynamics, oligomerization, and metal ion modulation. These findings pave the way for targeted strategies to regulate CLIC1 activity in pathological conditions, underscoring its potential as a therapeutic target.