CARMIL leading edge localization depends on a non-canonical PH domain and dimerization

CARMIL is a ~1370 amino acid cytoskeletal scaffold that plays crucial roles in cell motility and tissue development through interactions with cytoskeletal effectors and regulation of capping protein at the leading edge. However, the mechanism of CARMIL leading edge localization is unknown. Here we show that CARMIL interacts directly with the plasma membrane through its N-terminal region. The crystal structure of CARMIL(1-668) reveals that this region harbors a non-canonical pleckstrin homology (PH) domain connected to a 16 leucine-rich repeat domain. Lipid binding is mediated by the PH domain, but is further enhanced by a central helical domain. Small angle x-ray scattering reveals that the helical domain mediates antiparallel dimerization, properly positioning the PH domains for simultaneous membrane interaction. In cells, deletion of the PH domain impairs leading edge localization. The results support a direct membrane binding mechanism for CARMIL localization at the leading edge, where it regulates cytoskeletal effectors and motility.