Structural Insights into Membrane Interaction and Caveolar Targeting of Dynamin-like EHD2

The dynamin-related Eps15-homology domain-containing protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins. •Establishment of an EPR approach to study membrane interaction of EHD2•The helical domain of EHD2 is inserted into the membrane•The N terminus of EHD2 can switch from the G domain into the membrane•The N terminus regulates caveolar targeting of EHD2 EHD2 is a dynamin-like membrane-remodeling ATPase localizing to caveolae. Shah et al. show that residues at the tip of the helical domain can penetrate into the membrane. Amino-terminal residues switch from a hydrophobic pocket in the G domain into the membrane and regulate caveolar targeting of EHD2.