The SH3 binding site in front of the WH1 domain contributes to the membrane binding of the BAR domain protein endophilin A2.

The Bin-Amphiphysin-Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott-Aldrich syndrome protein (N-WASP). Here, we re-examined the binding sites of the SH3 domain of endophilin in N-WASP by machine learning-based prediction and identified the previously unrecognized binding site. In addition to the well-recognized PRM at the central proline-rich region, we found a PRM in front of the N-terminal WASP homology 1 (WH1) domain of N-WASP (NtPRM) as a binding site of the endophilin SH3 domain. Furthermore, the diameter of the membrane tubules in the presence of NtPRM mutant was narrower and wider than that in the presence of N-WASP and in its absence, respectively. Importantly, the NtPRM of N-WASP was involved in the membrane localization of endophilin A2 in cells. Therefore, the NtPRM contributes to the binding of endophilin to N-WASP in membrane remodeling.