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 v arious points close to the head-tail junction and measured its effects on the temperature dependence of velocity in microtubule gliding assa ys. The insert comprised two contiguous alpha-actinin triple coil repe ats 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 junc tion, at Asn(401) in the dimeric K partial derivative 401GST, the inse rt had no effect. When inserted closer to the heads at Val(376) into K partial derivative 376GST, the insert slowed progress below 22 degree s C but accelerated progress to similar to 125% of wild type above 22 degrees C. The most dramatic effect of the synthetic lever occurred wh en it was inserted very close to the head-neck junction, at Glu(340) i nto the single-headed construct K partial derivative 340GST. This cons truct was immotile without the insert, but motile with it, at about 30 % of the velocity of the dimeric control. The alpha-actinin module thu s confers some gain of-function when inserted close to the head-neck. junction but not when placed distal to it. The data exclude the presen ce of a lever arm C-terminal to Val(376) in the kinesin tail but sugge st that a short-throw lever arm may be present, N-terminal to Val(376) and contiguous with the head-neck junction at Ala(339).