Systematic Structural Biology of Dynein

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Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicular trafficking, ciliary/flagellar motility and cell division. These enzymes are highly complex containing up to thirteen different polypeptide components with a total mass of almost 2 MDa. In order to achieve a molecular understanding of mechanism, it will be essential to obtain high resolution structural information for all components of the dynein complex. However, the complexity and size of the dynein particle combined with the inherent flexibility of several domains has made obtaining high resolution structural information for the entire enzyme unfeasible. This application proposes a systematic approach to dynein structural biology by obtaining information for individual proteins or subdomains using multidimensional NMR techniques. These structures will then be used to build ever more complete models of the dynein motor and will enable structure-based hypotheses concerning function to be made. The specific aims for this submission center on analysis of dynein light chains and include: 1) molecular dynamics and refinement of the solution structure of LC1 which is associated with the dynein motor domain; 2) structural analysis of mutant forms of LC1 to allow interpretation of mutant phenotypes at the structural level; 3) completion of the NMR assignments and calculation of the solution structure for the Tctex1 light chain; 4) NMR structure analysis of thioredoxin-related and Ca2+-binding light chains; 5) preparation of other dynein domains for structure analysis following a systematic search for appropriate solution conditions. These studies will provide structure-based insight into the mechanisms of dynein regulation and will provide the necessary background for further structural analysis of this massive molecular motor.