DESCRIPTION (applicant's description) Dynein is one of the fundamental molecules that drive cytoskeletal-based eukaryotic cell movement. Virtually nothing is known regarding the structural details by which this motor utilizes an ATP hydrolysis mechanism to generate movement along a microtubule. This lack of knowledge contrasts sharply with information on kinesin and myosin, and represents a tremendous gap in our understanding of how chemical energy is used to produce movement. This application presents three specific aims that focus on structural determinations within a cytoplasmic dynein motor domain. Specific Aim One will utilize a cryo-electron microscopy approach to produce the first detailed three-dimensional model of a dynein motor domain. Experiments are described that will identify the locations of important functional domains within the motor and will begin to resolve the conformational changes that dynein undergoes as a result of nucleotide hydrolysis. The work will also investigate how dynein fits onto a microtubule and will identify the essential structural changes involved with movement. Specific Aim Two will determine the atomic structure for the dynein microtubule-binding domain. A major mutagenesis effort will also target the function of individual amino acids that bind dynein to a microtubule. This work will demonstrate how dynein interacts with a microtubule and how its affinity is modulated during the nucleotide hydrolysis cycle. Specific Aim Three seeks to determine the atomic structure for the dynein motor domain. Our group will make essential contributions in developing and analyzing the necessary motor domain fragments for a major crystallization effort. Success here will represent a landmark achievement in the field of cell motility. Each one of these aims will provide important and novel information on how the dynein motor works. Taken together, the aims represent a very powerful attack on a fundamental molecule of cell movement, and our laboratory is uniquely positioned to carry out this work. Success of these projects will not only pioneer an understanding of how dynein functions, but will also allow detailed comparisons with myosin and kinesin. All three families of motor proteins are essential for movement, development, and function of eukaryotic organisms. Defects in their activity lie at the root of a number of developmental and neurological diseases, and cancer.