What kinesin does at roadblocks: the coordination mechanism for molecular walking

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide‐induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide‐induced monomer (rK340) unbinding was 73.6 s −1 for ATP and 40.5 s −1 for ADP. Control ADP‐induced dimer (rK430) unbinding was 18.6 s −1 . Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP‐induced trail head unbinding was then 42 s −1 . This is less than two‐fold slower than the stepping rate of unimpeded rK430 dimers (50–70 s −1 ), indicating that during walking, lead head attachment induces at most only a slight (less than two‐fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s −1 ) ATP‐induced attachment of the lead head, followed by slower, strain‐sensitive ADP release from the lead head.