Mechanisms of CARMIL dimerization, autoinhibition, and capping protein binding

Capping protein (CP) regulates actin-based motility by blocking monomer exchange at the filament barbed end. Several proteins, including CARMIL, bind CP and allosterically weaken its affinity for the barbed end. CARMIL comprises pleckstrin homology (PH), leucine-rich repeat (LRR), helical dimerization (HD), CP-binding region (CBR), and proline-rich (PR) domains, but their roles in CP regulation remain unclear. We show that CARMIL1 is partially autoinhibited, with CBR (CARMIL1 961–1046 ) displaying greater uncapping activity than CARMIL1 1–1046 . A structure of CP-bound CARMIL1 1–1046 reveals a dimeric assembly, with PH-LRR on a plane flanked by HD and CBR-bound CP. A motif connecting HD to CBR-CP, the “antenna,” binds at the dimer LRR-LRR interface. An antenna mutant disrupting this interaction partially relieves autoinhibition in vitro. In CARMIL1 knockout cells, expression of the antenna mutant increases cell area, while deleting the myosin-I–binding PR domain induces membrane spikes. The results inform mechanisms of CARMIL dimerization, autoinhibition, and coordination of CP and myosin-I activities in cells.