| Eukaryotic cells use a diverse toolbox of cytoskeletal motors to transport and position cellular materials in space and time. Two microtubule-based motors---kinesin and dynein---transport organelles, RNA and protein cargos over long-distances. While multiple kinesin motors are used for long-distance plus- end-directed transport, a single type of dynein---cytoplasmic dynein 1--- performs nearly all minus-end-directed tasks. Despite cytoplasmic dynein's role in such diverse activities, many aspects of its molecular mechanism remain poorly understood. My thesis work uses a combination of cryo-electron microscope (EM) structural biology and single-molecule approaches to provide novel insights into the mechanistic basis of how dynein interacts with its microtubule track and how microtubule binding is regulated by the ubiquitous co-factor, Lis1.;First, we solved a 9.7A structure of dynein's microtubule binding domain bound to microtubules. This structure allowed us to identify large conformational changes that occur in dynein's microtubule-binding domain upon track binding. We hypothesize that these conformational changes allosterically regulate the ATP hydrolysis cycle in dynein's motor domain, which is located over 25 nm from the site of microtubule binding. Molecular dynamics simulations, followed by single-molecule assays, allowed us to identify dynamic salt bridge switches in dynein, which can tune its affinity for the microtubule. The native dynein, which has been selected for submaximal processivity, might allow a broader dynamic range for regulation.;Second, we identified how dynein is regulated by its ubiquitous co-factor, Lis1. Our three-dimensional cryo-EM structures of the dynein-Lis1 complex showed dynein's mechanical element, the linker, is in an altered position in the presence of Lis1. Fluorescence resonance energy transfer (FRET) and single- molecule studies indicated that Lis1 binding to dynein sterically blocks the dynein linker from reaching its normal docking site, which may interrupt dynein's mechanochemical cycle and prevent its release from microtubules. |
