A Mechanical Switch from Diffusion to Directional Motion Activates ATPase in Dynein Motor

Abstract

Dynein is a motor protein that moves along microtubule tracks via the energy from ATP hydrolysis. Unlike other processive cytoskeletal motors, the dynein step size is highly variable with a significant level of diffusion. To investigate the molecular basis of the stochastic nature of dynein stepping, we here characterized the structure, physical properties, and effects of site-directed mutations of the dynein-microtubule interface.We found that mutation of either the R403 or E416 residue of α-tubulin to alanine changed the directional movement of the microtubules on a dynein-coated surface to undirected thermal diffusion, resulting in a loss of dynein ATPase activity. Biochemical and cryo-electron microscopy analyses of the microtubule binding domain (MTBD)-microtubule complex revealed that these tubulin residues switch dynein from diffusional to stationary binding by forming salt bridges with the residue in H1 and H6 of the MTBD. The formation of two salt bridges then triggers a registry change in the stalk coiled coil required for ATPase activation, and thus leads to directional movement. In this mechanism, the previously undescribed interaction between α-R403 and E3390 in H1 of the MTBD plays a key role, and is likely to explain the fact that the equivalent tubulin mutation in mammals (R402) can cause lissencephaly (Keays et al., Cell 128, 45-).Compared to kinesin-microtubule interactions, where the weak-to-strong state transition is mediated by several contact sites involving a few tubulin residues (Uchimura et al., EMBO. J., 29, 1167-), for dynein, the mechanical switch from diffusional to stationary binding is controlled by only two salt bridges. Because of this pinpoint regulation, the stepping motion of dynein might be only loosely coupled with the reaction of ATP hydrolysis, resulting in the variable step size.