Chai, P.; Yang, J.; Geohring, I. C.; Markus, S. M.; Wang, Y.; Zhang, K.

Dynein-mediated cargo transport plays pivotal roles in a variety of essential cellular activities, including vesicle trafficking, organelle and nucleus positioning, mitosis and morphogenesis. Central to all these cellular processes is dynein's ATP-dependent mechanochemical cycle. Here, we perform a systematic cryo-electron microscopic investigation of the conformational landscape of full-length human dynein-1 actively undergoing nucleotide-induced conformational changes, both on and off microtubules. Our approach reveals over 30 high-resolution structures, categorized into nine distinct states of the AAA+ motor ring. This provides a dynamic and comprehensive view of dynein throughout its mechanochemical cycle. The novel intermediate states unveil a series of important mechanistic insights into dynein function, including a 'backdoor' phosphate release mechanism that coordinates linker straightening, a stepwise structural basis for the bidirectional communications between ATP binding/hydrolysis and microtubule-binding affinity, and elucidate the mechanism by which microtubule binding increases ATPase activity. Our findings also lead to a substantially revised model for the force-generating power stroke, clarifying the means by which dynein achieves its unidirectional stepping on microtubules. These results substantially enhance our understanding of dynein function and provide a more complete model of its mechanochemical cycle.