UCSF

Cytoplasmic dynein, a microtubule-based motor protein, transports many intracellular cargos by means of its light intermediate chain (LIC). However, the mechanism by which the LIC connects cargo to dynein has largely been unknown. Here in this thesis, I describe two crystal structures: the conserved region of the dynein light intermediate chain from a fungal species and the LIC-binding domain of the human dynein cargo adaptor Hook3. Despite its structure having a GTPase-like fold, fungal LIC has lost its ability to bind nucleotide, while human LIC1 binds GDP preferentially over GTP. We show that the LIC G domain binds the dynein heavy chain using a conserved patch of aromatic residues, whereas the less conserved C-terminal domain binds several Rab effectors involved in membrane transport, including Hook3. By solving the crystal structure of Hook3's LIC-binding domain and using structure-based mutagenesis, we identify two conserved surface residues that are each critical for the adaptor’s ability to interact with LIC1.We also show that the LIC1-Hook3 interaction is vital for assembling the entire dynein-dynactin complex.In addition, using a series of Hook3 constructs, we have been able to dissociate dynein-dynactin-adapter complex formation from the activation of motility. This result suggests that there is an additional allosteric activation step beyond complex assembly. This doctoral work not only provides insight into how the LIC binds both the dynein complex and cargo, but it also opens questions for the GTPase field and dynein motility.