A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) O f Drosophila kinesin heavy chain is identical in all superfamily members an d is located in alpha-helix 5 on the microtubule-binding surface of the cat alytic motor domain. Substitution of methionine at Thr(291) results in comp lete loss of function in vivo. In vitro, the T291M mutation disrupts the AT Pase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D, J,, Gilbert, S. P,, and Saxton, W, M, (1999) J, Biol. Chem , 274, 31506-31514), The pre-steady-state kinetic analysis presented here s hows that ATP binding is weakened significantly, and the rate ofATP hydroly sis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The resuits indicate that there is a signaling defect betwee n the motor domains of the T291M dimer, The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more i ndependently rather than in the strict, coordinated fashion that is typical of kinesin.