Abstract
A power stroke of dynein is thought to be responsible for the stepping of dimeric dynein. However, the actual size of the displacement driven by a power stroke has not been directly measured. Here, the displacements of single-headed cytoplasmic dynein were measured by optical tweezers. The mean displacement of dynein interacting with microtubule was ~8 nm at 100 µM ATP, and decreased sigmoidally with a decrease in the ATP concentration. The ATP dependence of the mean displacement was explained by a model that some dynein molecules bind to microtubule in pre-stroke conformation and generate 8-nm displacement, while others bind in the post-stroke one and detach without producing a power stroke. Biochemical assays showed that the binding affinity of the post-stroke dynein to a microtubule was ~5 times higher than that of pre-stroke dynein, and the dissociation rate was ~4 times lower. Taking account of these rates, we conclude that the displacement driven by a power stroke is 8.3 nm. A working model of dimeric dynein driven by the 8-nm power stroke was proposed.
Highlights
The size of the power stroke has been investigated by the structural studies[7,8,9,10,11]
To detect the power stroke of dynein, we constructed D384GB, in which BFP and GFP were fused to the AAA2 domain and the N-terminus of the linker in D384, respectively (Fig. 1A and C)
The step was driven by the power stroke because the mutant without linker swing did not take steps
Summary
The size of the power stroke has been investigated by the structural studies[7,8,9,10,11]. In the absence of microtubules, the position of the microtubule binding domain (MTBD) relative to the tail domain of dynein was changed by ~15 nm from the ADP-Vi to the apo states[7,8] (Table S1). Three-dimensional electron microscopy structures of axonemal dynein in axoneme were reconstructed, and the ring domain and MTBD of dynein were shifted by 5–12 nm relative to the tail domain along the microtubule from the prestroke to poststroke states[9,10,11] (Table S1). To understand the mechanochemical reaction of human cytoplasmic dynein, we investigated the displacement of microtubules driven by the power stroke of a single-headed dynein by optical tweezers. The ATP dependence of the mean displacement was explained by a mechanochemical model. From the results of 8-nm displacement and the chemical rates, we proposed a walking model of dimeric dynein
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