Structure-Function Studies of Loop L5 in the Mitotic Kinesin Eg5

Abstract

All processive kinesins contain an unusual structural motif consisting of an alpha helix (α2) interrupted in the middle by a stem and loop motif known as L5. The role of L5 in the overall enzymatic function of kinesin motors remains unknown. However, its importance is highlighted by the finding that a variety of small molecule inhibitors of the mitotic kinesin Eg5, which contains the longest L5 in the kinesin superfamily, bind to this region with high affinity. These inhibitors induce a folding of L5, and trap the motor in an ADP bound, weak microtubule binding conformation. In order to gain greater insight into the function of L5, we have characterized three site-directed mutants in the loop (position 121), stem (position 131) or in the portion of α2 amino-terminal to this stem and loop motif (position 113). These three mutations have profound effects on the kinetics of structural transitions that occur with nucleotide and microtubule binding, and our results indicate that this conserved structural motif plays an important role in tuning the kinetics of kinesin motors.