The central α-helical domain as a key regulatory module in Moesin activation revealed by single-molecule fluorescence

Moesin is a member of the ERM (Ezrin-Radixin-Moesin) protein family that links the plasma membrane to the actin cytoskeleton through a conformational activation process. While autoinhibition is known to be regulated by interactions between the N-terminal FERM domain and the C-terminal domain (CTD), the role of the central α-helical domain (CHD), which connects these regions, remains poorly understood. Here, we combine single-molecule and ensemble Förster resonance energy transfer (FRET) with complementary biophysical approaches, including circular dichroism spectroscopy and isothermal titration calorimetry, to investigate the intrinsic conformation, stability, and ligand responsiveness of the CHD. Single-molecule FRET measurements reveal that the CHD adopts a compact helical bundle that is intrinsically stable and independent of FERM-CTD interactions, a finding further supported by thermal denaturation analysis. Upon binding of phosphatidylinositol 4,5-bisphosphate (PIP2), both single-molecule and ensemble FRET analyses show a pronounced increase in intradomain distances, indicating a transition from a compact bundle to a more extended conformation. Consistently, isothermal titration calorimetry confirms direct binding between PIP2 and the CHD. Together, these results identify the CHD as an active regulatory element, rather than a passive linker, and suggest that its conformational remodeling provides a critical structural basis for Moesin activation. More broadly, this work offers new mechanistic insight into how lipid binding is coupled to large-scale conformational transitions in ERM proteins.