Crystal and EM Structures Reveal a Mechanism for Long-Range Allosteric Communication in the Yeast Dynein Motor Domain

Abstract

Dynein, a member of the AAA family of ATPases, “walks” toward the minus end of microtubules using energy from ATP hydrolysis. Recent atomic structures of the dynein motor domain with no nucleotide (apo; yeast) or ADP (Dictyostelium) in the main hydrolytic site (AAA1) have begun to yield insights into how this fascinatingly complex machine may work. Here, we have solved the crystal (3.5 A) of the yeast dynein motor domain with a nonhydrolyzable nucleotide (AMPPNP) bound at AAA1. By comparison to the prior apo structures of yeast dynein, the new structure reveals that AMPPNP causes a large conformational change of AAA domains in the dynein ring in which AAA2 moves closer to AAA1. The linker, the proposed mechanical element of dynein, remains largely in a similar conformation with its N-terminal docked onto AAA5, but new interactions are made with AAA2. Further EM analysis of dynein bound to several ATP analogs showed that the linker remains docked on the ring in all states, with a small conformational change observed when bound to the analog ATP-vanadate. Mutagenesis studies show that disruption of a single salt-bridge between the linker and AAA2 blocks microtubule stimulation of AAA1 ATPase activity and nucleotide-induced changes in microtubule binding affinity. Collectively, our data reveal a new nucleotide triphosphate bound state of the dynein motor domain and suggest that the linker serves not only in dynein mechanics but also plays a critical role in the long range allosteric communication between dynein's main ATPase site and the microtubule binding domain.