Structural Basis for the Regulation of Microtubule-Binding Affinity by Cytoplasmic Dynein

Abstract

The molecular motor cytoplasmic dynein is responsible for most minus-end directed microtubule-based transport in eukaryotic cells. Dynein is especially important in neurons, where defects in dynein-based motility have been linked to neurodegenerative and neurodevelopmental diseases in humans. Due to the complexity and size of dynein our understanding of it has lagged behind that of other cytoskeletal motor proteins. A major goal in the field is to determine how ATP hydrolysis events in dynein's AAA+ motor domain “ring” are communicated to the microtubule-binding domain (MTBD), which is located 250A away. Information between these two sites is proposed to be conveyed via changes in the register of a long coiled-coil “stalk” that connects the MTBD to the AAA+ ring. Both low and high affinity registers have been identified and a crystal structure of the MTBD with the stalk locked into the low affinity register has been solved. To understand the structural changes that occur upon MT binding and how they might be regulated we have obtained a ∼10A resolution cryo-EM reconstruction of microtubules decorated with the MTBD of cytoplasmic dynein locked into the “high affinity” state. We used our density to perform molecular dynamics flexible fitting with the available crystal structure and observe significant rearrangements in three of the six alpha helices that make up the MTBD. Our pseudo-atomic model accounts for several mutations that were previously identified for their effects on microtubule-binding affinity. Based on our results we propose a model for the coordination between changes in the register of the coiled-coil and dynein's affinity for its track.