A torque component in the kinesin-1 power stroke Junichiro Yajima & Robert A Cross Kinesin-1 is a twin-headed molecular motor that moves along microtubules in 8-nm steps, using a walking action in which the two heads interact alternately with the microtubule 1–4 . Constructs with only one head can also produce impulses of force and motion 5–7 , indicating that the walking action is an amplification strategy that leverages an underlying force- generating event. Recent work suggests that directional force is produced either by directionally biased selection of microtubule binding sites 8,9 or by a conformational change subsequent to the binding event 10–12 . We report here that surface-attached rat kinesin-1 monomers drive counterclockwise rotation of sliding microtubules around their axes, and that by manipulating the assay geometry, we could reduce or block the torsional motion with negligible effects on the axial motion. We can account for this behavior on the simple assumption that kinesin heads tend to bind to the closest available tubulin heterodimer in the lattice, but only in the case where an additional biasing process is present that shifts the start position for diffusion-to-capture toward the microtubule plus end by B1 nm. Using a specialized microtubule sliding assay in which the microtu- bules carry a side arm, we discovered that microtubules sliding across lawns of single-headed rat kinesins (RK340Gel; a fusion to gelsolin) undergo short-pitch rotation around their longitudinal axes, as revealed by the shifting orientation of the side arms (Fig. 1; Supple- mentary Video 1 online). This short-pitch rotational motion has not been previously seen for kinesin-1, and it demonstrates that kinesin-1 single heads produce torsional force as well as axial sliding force. In many past experiments, microtubule rotation was counterclock- wise relative to an observer looking along the axis toward the microtubule minus end. The microtubule side arms tended transiently to block rotation when they collided once per rotation with the coverslip surface. During these episodes, microtubule sliding contin- ued while torque built up until it was sufficient to drive the side arm around underneath the microtubule in a sudden release of the torsional load. The rotational pitch was 0.48 ± 0.11 mm (mean pitch ± s.d.; Fig. 2a, Table 1). The rotation rate did not vary for side arms of different length (0.5 mm–1.5 mm), provided that the ratio of the side- arm length to the length of the main part did not exceed 0.3. The rotation of microtubules while sliding has implications for the role of weak binding in the mechanochemical kinetic cycle of kinesin-1. ATP turnover drives kinesins in general to cycle between an ADP-containing weak binding state, which is unstably attached to the microtubule, and a series of strong binding states, which are stably attached and can hold force 13,14 . The weakly bound, ADP-containing state tends to dominate, even at high concentrations of ATP 15,16 . Our data imply that the weakly attached heads, despite their numerical dominance, are unable to oppose the rotational force produced by a few strongly bound heads. What sets the rotation rate? We found that the rotational pitch was insensitive to microtubule geometry. Twelve-protofilament microtu- bules have helically arranged protofilaments with a 3–4 mm (right- handed) supertwist, whereas 13-protofilament microtubules have Streptavidin Biotinylated DNA RK345-Linker-Sp1 a b Gelsolin RK340 Bent microtubule Bent microtubule 14.4 13.4 12.6 11.7 11.1 7.5 6.5 5.3 3.9 0.0 14.2 13.2 10.8 6.8 2.8 1.3 1.0 0.0 16.5 19.8 21.4 22.4 23.0 23.6 24.6 26.7 Figure 1 Single-headed kinesins induce microtubules to rotate while sliding. (a) Two complete rotations of a side-armed microtubule sliding on an RK340Gel surface. In the cartoon and the data, the microtubule is sliding in the arrowed direction and rotating counterclockwise relative to an observer looking along the microtubule in the direction of motion. (b) Two complete rotations of a side-armed microtubule sliding on a streptavidin/biotinylated DNA/Sp1/RK345 ‘sandwich.’ Bar, 3 mm. Supplementary Videos 1 and 3 illustrate the rotation. Received 18 July; accepted 20 September; published online 9 October 2005; doi:10.1038/nchembio740 Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, UK. Correspondence should be addressed to R.A.C. (r.cross@mcri.ac.uk). 338 VOLUME 1 NUMBER 6 NOVEMBER 2005 NATURE CHEMICAL BIOLOGY LETTERS © 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology