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.