Single-Molecule Study of the Communication between the Two Primary Sites of Cytoplasmic Dynein

Cytoplasmic dynein is the primary minus-end directed microtubule (MT) motor in eukaryotic cells. The motor domain of dynein consists of six AAA+ subunits, forming a ring. Previous studies showed that AAA1 is the main site of ATP hydrolysis, responsible for force generation and MT detachment. The AAA3 site is also required for robust motility, but its role in dynein's mechanochemical cycle is unknown. In this study, we use single-molecule fluorescence to elucidate the role of AAA3 and its interactions with AAA1 during stepping. We find that AAA3 regulates MT detachment. AAA3 mutants of dynein are gated by MT release and AAA1 requires timely detachment to proceed through its own hydrolysis cycle. High-resolution fluorescence tracking indicated that substrate release from AAA1 is rapid compared to catalysis, consistent with the open conformation of AAA1 observed in crystal structures. Consistent with loose substrate binding at AAA1, the catalytic activity of AAA3 can be specifically inhibited by a nonhydrolyzable ATP analog at low concentrations. Analysis of analog-induced pause density indicates that two analogs are required to initiate pausing, and that the analogs bind in an uncoordinated manner. By analyzing the pausing behavior at different velocities and at different inhibitor concentrations, we gain insight into the nature of communication between AAA1, AAA3, and the MT-binding domain. On the basis of our results, we propose a model for the role of AAA3 in dynein's stepping mechanism.