Small Crowders Slow Down Kinesin-1 Stepping by Hindering Motor Domain Diffusion

The dimeric motor protein kinesin-1 moves processively along microtubules against forces of up to 7 pN. However, the mechanism of force generation is still debated. Here, we point to the crucial importance of diffusion of the tethered motor domain for the stepping of kinesin-1: small crowders stop the motor at a viscosity of 5 $\mathrm{m}\mathrm{P}\mathrm{a}·\mathrm{s}$—corresponding to a hydrodynamic load in the sub-fN ($\sim 10^{-4}\mathrm{pN}$) range—whereas large crowders have no impact even at viscosities above 100 $\mathrm{m}\mathrm{P}\mathrm{a}·\mathrm{s}$. This indicates that the scale-dependent, effective viscosity experienced by the tethered motor domain is a key factor determining kinesin’s functionality. Our results emphasize the role of diffusion in the kinesin-1 stepping mechanism and the general importance of the viscosity scaling paradigm in nanomechanics.

Figure 1

Experimental setup of kinesin-1 stepping assays, drawn to scale. Hydrodynamic radii of the different PEGs are 2.17 nm, ($6\mathrm{kg}/\mathrm{mol}$), 3.85 nm ($18\mathrm{kg}/\mathrm{mol}$), and 35 nm ($1000\mathrm{kg}/\mathrm{mol}$).

Figure 2

Velocity of kinesin-1 motion along the MTs measured in stepping motility assays plotted against the following: (a) macroscopic solution viscosity and (b) effective viscosity experienced by a kinesin-1 motor domain [Eq. (1)], normalized by pure buffer viscosity. Error bars are standard deviation of the mean. Solid line is a fit of the load-dependent stepping model by Schnitzer et al. [42] (details in the text). Legend refers to both panels.

Figure 3

Mechanism of kinesin-1 stepping [24]. The motor spends most of the time in the ATP-waiting state, $\mathbf{1}$. Binding of ATP to the MT-bound motor domain changes its conformation, enhancing the orientational freedom of the neck linker. The $\mathbf{1}\to \mathbf{2}$ reaction is reversible. As long as the MT-bound motor domain is in the ATP-bound state, the tethered domain can reach the subsequent binding site on the MT and release ADP. This movement, realized by diffusion, is constrained by viscosity and tension arising in the neck linker. When ATP is hydrolyzed, the MT-bound domain detaches from the MT, becoming the tethered one ($\mathbf{3}\to \mathbf{1}$).