Engineering a lever into the kinesin neck

To probe for a lever arm action in the kinesin stepping mechanism, we engineered a rodlike extension piece into the tail of rat kinesin at various points close to the head-tail junction and measured its effects on the temperature dependence of velocity in microtubule gliding assays. The insert comprised two contiguous alpha-actinin triple-coil repeats and was predicted to fold into a stiff rodlike module about 11 nm long. The effects of this module were greater the closer it was placed to the head-tail junction. When inserted distal to the head-tail junction, at Asn401 in the dimeric K partial differential401GST, the insert had no effect. When inserted closer to the heads at Val376 into K partial differential376GST, the insert slowed progress below 22 degreesC but accelerated progress to approximately 125% of wild type above 22 degreesC. The most dramatic effect of the synthetic lever occurred when it was inserted very close to the head-neck junction, at Glu340 into the single-headed construct K partial differential340GST. This construct was immotile without the insert, but motile with it, at about 30% of the velocity of the dimeric control. The alpha-actinin module thus confers some gain-of-function when inserted close to the head-neck junction but not when placed distal to it. The data exclude the presence of a lever arm C-terminal to Val376 in the kinesin tail but suggest that a short-throw lever arm may be present, N-terminal to Val376 and contiguous with the head-neck junction at Ala339.