The Structural Basis for Microtubule Binding and Release by Dynein

Eukaryotic cells face a considerable challenge organizing a complicated interior with spatial and temporal precision. They do so, in part, through the deployment of the microtubule-based molecular motors kinesin and dynein, which translate chemo-mechanical force production into the movement of diverse cargo. Many aspects of kinesin's motility mechanism are now known in detail, whereas fundamental aspects of dynein's motility mechanism remain unclear. An important unresolved question is how dynein couples rounds of ATP binding and hydrolysis to changes in affinity for its track, a requisite for a protein that takes steps. Here we report a sub-nanometer cryo-EM reconstruction of the high affinity state of dynein's microtubule binding domain in complex with the microtubule. Using molecular dynamics flexible fitting, we determined a pseudoatomic model of the high affinity state. When compared to previously reported crystal structure of the free microtubule binding domain, our model revealed the conformational changes underlying changes in affinity. Surprisingly, our simulations suggested that specific residues within the microtubule binding domain may tune dynein's affinity for the microtubule. We confirmed this observation by directly measuring dynein's motile properties using in vitro single molecule motility assays, which demonstrated that single point mutations of these residues dramatically enhance dynein's processivity. We then sought to understand why dynein has been selected to be a restrained motor, and found that dynein-driven nuclear oscillations in budding yeast are defective in the context of highly processive mutants. Together, these results provide a mechanism for the coupling of ATPase activity to microtubule binding and release by dynein, and the degree to which evolution has fine-tuned this mechanism. I conclude with a roadmap of future approaches to gain further insight into dynein's motility mechanism, and describe our work developing materials and methods towards this goal.