Engineering the processive run length of the kinesin motor

Conventional kinesin is a highly processive molecular motor that takes seve ral hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two h eads of the kinesin dimer, but the specific factors that determine kinesin' s run length (distance traveled per microtubule encounter) are not known. H ere, we show that the neck coiled-coil, a structure adjacent to the motor d omain, plays an important role in governing the run length. By adding posit ive charge to the neck coiled-coil, we have created ultra-processive kinesi n mutants that have fourfold longer run lengths than the wild-type motor, b ut that have normal ATPase activity and motor velocity. Conversely, adding negative charge on the neck coiled-coil decreases the run length. The gain in processivity can be suppressed by either proteolytic cleavage of tubulin 's negatively charged COOH terminus or by high salt concentrations. Therefo re, modulation of processivity by the neck coiled-coil appears to involve a n electrostatic tethering interaction with the COOH terminus of tubulin. Th e ability to readily increase kinesin processivity by mutation, taken toget her with the strong sequence conservation of the neck coiled-coil, suggests that evolutionary pressures may limit kinesin's run length to optimize its in vivo function.