Engineered Dynein Mutants Reveal Minimal Structural and Catalytic Requirements for Processive Motility

Abstract

Cytoplasmic dynein is a molecular motor responsible for minus-end directed transport along microtubules. In contrast to kinesins and myosins, the detailed mechanism of dynein processivity and force generation remains unclear. Dynein’s structure and evolutionary origin are different from these motors, suggesting unique mechanistic features. In this work, we engineered novel dynein constructs with altered mechanical, chemical and geometric properties to test the roles of rigid linkage between monomers, interaction between the ATPase rings and the proposed linker swing mechanism in maintaining dynein processivity. We found that a rigid linkage between monomers and dimerization through the N-terminal tail domains are not essential for dynein processivity. Instead, processivity minimally requires the linker domain of one monomer with an active ATPase ring to be attached to a partner monomer, which can be replaced by an inert protein retaining the microtubule-binding domain. To understand how one active head could provide motility to an inactive partner, we quantified the force-dependent microtubule release rates of dynein monomers. A clear directional asymmetry for detachment was observed, with significantly faster release towards the minus-end, providing insight into dynein’s directionality. These results led us to a detailed mechanistic model of dynein processivity, directionality and force generation.