A structural change in the neck region of kinesin motors drives unidirectional motility.

摘要

Kinesin motors power several intracellular processes by converting energy from ATP hydrolysis into motion along microtubules. Kinesin walks unidirectionally towards the plus ends of microtubules, can processively walk hundreds of steps without dissociating, and is about 50% efficient in converting the chemical energy of ATP into the mechanical energy of motility. The mechanism by which kinesin could accomplish this was unknown.; To investigate whether conformational changes in the kinesin neck linker (the first 10 amino acids of the neck) drive the motor's unidirectional forward motion, my collaborators and I did EPR and FRET spectroscopy as well as undecagold electron microscopy on the kinesin neck linker. The neck linker undergoes a disordered to ordered transition, zipping up onto the catalytic core, when the motor binds ATP. When the motor releases phosphate, the neck linker returns to its original disordered state. This rearward to forward conformational change drives kinesin's plus-end directed motility on microtubles.; We tested whether the neck linker conformational change was linked to the kinesin dimer's coordinated, processive motility by examining whether two ATP nonhydrolyzing mutants, G234A and E236A, could undergo this conformational change. While defective in ATP hydrolysis, an E236A kinesin dimer had coordinated ADP release of its two heads and was able to undergo the neck linker conformational change. The G234A dimer, however, could not coordinate ADP release between its two heads, and did not exhibit a conformational change in its neck linker between the ATP and ADP states. This result showed that this conformational change is critical to head-head coordination in the kinesin dimer.; We characterized the thermodynamics of the neck linker conformational change using EPR spectroscopy. In the presence of triphosphate nucleotides and microtubules, the free energy change between the docked and undocked states of the neck linker is small, whereas the enthalpy and entropy changes are very large. One would expect this for a disordered to ordered transition. The large entropic cost is balanced out by a large favorable enthalpy change. Furthermore, the small free energy change seen between docked and undocked states of the neck linker may explain why kinesin is extremely efficient.