The movement of a molecular motor protein along a cytoskeletal track requires communication between the ATPase and polymer-binding sites. While these sites are located in close proximity in kinesin and myosin, dynein has a ~135 Å coiled-coil stalk that separates the microtubule-binding domain (MTBD) from the AAA ATPase ring. An analysis of 534 unique dynein sequences revealed that the length of the stalk is highly conserved. A panel of mutants based on our analysis revealed three regions of the

more »

... stalk for which altering the length resulted in diffusional motion along microtubules. Two diffusive mutants also showed hyperactive ATPase activity, and were no longer sensitive to microtubules. Structural analysis of one of these mutants using cryo electron microscopy revealed a previously uncharacterized open conformation of the AAA ring. This conformation is lowly populated in the wild-type protein and thus may be an on-pathway catalytic intermediate. Our results reveal how changes in the stalk can affect the conformation and activity of the AAA ring, and modulate dynein motility.