Direct Observation Of The Allosteric Conformational Change Of Kinesin-1 Using Gold Nanorod And Its Implication For Head-Head Coordination

Kinesin-1 is a motor protein that moves along microtubules by alternately moving two motor domains (heads) in a hand-over-hand manner. The neck linker, short stretch that connects two heads, has been shown to essential for the coordination between heads, although the underlying structural basis is unknown. Recently solved crystal structure revealed a subdomain rotation that takes place upon ATP hydrolysis; closing the nucleotide-binding pocket and opening a hydrophobic pocket at the opposite side, which will be filled with the neck linker. We hypothesized that this subdomain rotation is required for promoting ATP hydrolysis but is suppressed in the leading head because backward tension prohibits the docking of the neck linker. Here we tested this hypothesis by directly observing the rotational motion of the subdomain of one of kinesin heads during processive movement. We observed gold nanorods specifically attached to one of the heads using dark-field microscopy and determined its angles and positions at 100 μs temporal resolution and one-degree angle precision (Enoki et al. Anal. Chem. 2015). The angle of gold nanorod changed once while the labeled head binds to the microtubule and the angle reversed after detachment from microtubule. The dwell times in the pre- and post-rotation states were nearly equal at saturating ATP condition, indicating that the labeled head is in the pre- or post-rotation states when the head is in the leading or trailing positions, respectively. Mutation in the neck linker, which destabilizes the neck-linker docking, increased the dwell time in the pre-rotation state, supporting the idea that the neck-linker docking is essential for stabilizing the subdomain rotation. These results explain how the neck linker tension/orientation allosterically regulates ATP hydrolysis differently in the leading and trailing heads.