Dynein Adaptors Such as Drosophila Bicaudal (Dm BicD) Recognize Cargo and are Required to Activate Dynein for Processive Transport

Abstract

In the absence of cargo, auto-inhibited BicD cannot recruit dynein, but the underlying mechanism of cargo-induced activation is elusive. Our single-molecule processivity assays show that auto-inhibition is abolished in the full-length Bicaudal mutant F684I, which activates dynein motility even in the absence of cargo. To investigate the structural basis for activation, we determined the X-ray structure of the C-terminal cargo-binding domain (CTD) of Dm BicD-CTD/F684I at 2.4 Å resolution. The structure revealed that the mutant has a homotypic coiled-coil registry, in which the two helices of the homodimer are aligned, whereas the wild-type structure has an asymmetric registry, in which the two chains are vertically shifted by ∼1 helical turn in a portion of the coiled-coil. In the mutant, N- terminal ∼20-residue region is disordered for one of the two chains. MD simulation and CD spectroscopy data suggested the structural flexibility of N-terminal region of Dm BicD-CTD/F684I and confirmed the intact protein is in the crystal and alpha-helical. We propose that a coiled-coil registry shift activates BicD for dynein recruitment. The registry shift could either be induced by the F684I mutation or by cargo-binding. Also, the human homolog of the BicD-CTD/F684I mutant (BicD2-CTD/F743I) showed diminished binding to its cargo Nup358. Thus, we propose that in mammalia, a coiled-coil registry shift modulates cargo selection for BicD2-dependent transport pathways, which are important for brain development and chromosome segregation. Furthermore, we investigated the mechanism for cargo recognition by BicD2. Our data from Circular Dicroism spectroscopy and small-angle X-ray scattering suggest that the BicD2 binding domain of Nup358, which is intrinsically disordered on its own, takes on a helical structure when binding to BicD2. This alpha-helix of Nup358 could be a structural feature for cargo recognition by BicD2.