| Abstract
| - Strict coordination of the two motor domains of kinesin is required for driving the processivemovement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to becritical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164Kexhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules.Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled onthe microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu164, whichhas been conserved through evolution, is located at the motor−microtubule interface close to key residueson helix α12 of β-tubulin. We explored further the contributions of Glu164 to motor function using severalsite-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that themicrotubule−E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was ableto dissociate a population of E164K motors from the microtubule but could not dissociate E164A. Wetested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The resultsprovide new insights about the motor−microtubule interface and the pathway of communication forprocessive motility.
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