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Structural and Kinetic Mechanisms of the Yeast Cytoplasmic Dynein Motor Domain


Dynein is a cytoskeletal motor that drives minus-end directed transport on microtubules. Although more than 40 years have passed since dynein was first discovered the molecular basis of dynein's motility and force production remains to be elucidated. There is yet no high resolution structure of the motor domain, nor a clear picture of the steps in the kinetic cycle. The work presented here focuses on dissecting the kinetic and structural mechanisms of yeast cytoplasmic dynein motility. In Chapter 1, the role of ATP binding and hydrolysis in dynein's multiple AAA domains was investigated by single molecule motility and steady state kinetic assays of recombinant yeast cytoplasmic dynein. We found that AAA3 and AAA4 ATP hydrolysis mutants displayed decreased motility, force production, and ATPase activity, but did not abolish processivity. In addition, we found no kinetic evidence for multiple ATP hydrolyses during the ATPase cycle, suggesting that AAA3 and AAA4 might play roles in allosterically regulating the dynein motor. Chapter 2 provides a description of the structure of the yeast cytoplasmic dynein motor domain solved to ~6 angstroms by X-ray crystallography. We uncovered the structure of dynein's domains - the linker, AAA domains, and the stalk, as well as discovering a novel structure termed the buttress. From this structure, we proposed a AAA ring closure mechanism to explain how ATP binding at dynein's main ATPase site, AAA1, triggers allosteric changes in other parts of the motor. In Chapter 3, we provide a comparison of the crystal structure presented in Chapter 2 and a subsequent struture of the Dictyostelium dynein motor domain. From this analysis, we provide further support for AAA ring closure, as well as discussing the dimeric orientation of dynein on microtubules during processive motility.

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