Fig. S7.
Assembly of EHD4. (A) Crystal packing of ATPγS-bound EHD4ΔN, shown in two different views. The crystals are built of four EHD4 filaments, which are rotated 90° to each other (Left). In Right, alternate dimers are colored in blue and red (light/dark for the monomers). (B) Schematic arrangement of EHD4 dimers in the oligomer. At Right, the linear oligomer is shown from a side view. The distance between the first amino acid of EHD4ΔN (Gln22) and the membrane interacting Val359 (corresponding to Cys356 in EHD2; Fig. 2) is 42 Å. To allow the N-terminal residues to insert into the same membrane leaflet as the primary membrane binding site in the helical domain, additional structural rearrangements, such as a partial unfolding of helix α1 may be required. Our previously published EPR data (16) showed that spin labels attached to the N terminus of EHD2 experience highly increased accessibility to oxygen in the presence of membranes, indicating that the N terminus is immersing into the membrane in the presence of liposomes (hence its denotation as “secondary membrane binding site”). Furthermore, continuous wave measurements revealed that N-terminal residues display increased mobility upon membrane binding, consistent with a release of the N terminus in the presence of membranes. In general, the N-terminal residues in EHD proteins are hydrophobic and highly conserved in the EHD family, suggesting a conserved function. However, in a cellular context (e.g., at caveolar or endosomal membranes), the exact position of the N terminus relative to the primary membrane binding site in the helical domain is not known. (C) Surface conservation plot of EHD4, using 39 EHD sequences from 20 different species. Conserved residues are shown in purple and nonconserved residues in cyan. Note that the oligomerization interface between the GTPase domain and helical domain is highly conserved.