Affinity regulation mechanism of microtubule-binding domain of cytoplasmic dynein

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Cytoplasmic dynein is a motor protein that walks on microtubules (MTs). The microtubule-binding domain (MTBD) is located at the tip of the anti-parallel coiled-coil, which is protruded from the ATPase domain of dynein heavy chain. It has been considered that the change in the association mode of the coiled-coil alters the MT affinity of the MTBD. However, the structural mechanism underlying the affinity regulation of the MTBD remains elusive.Here, we designed two MTBD constructs, termed MTBD-High and MTBD-Low, which are stabilized either in the high or low affinity state for MTs, respectively, by introducing a disulfide bond between the coiled-coil helix. We performed NMR analyses of MTBD-High and MTBD-Low, in order to elucidate differences in the structure and the MT-binding mode in the high and low affinity states. Based on NMR data in combination with the cryoEM analysis of the MTBD-High/MTs complex, we modeled the complex structure of the MTBD and MTs in the strong binding state.