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.
Crevel
Isabelle M-T C
IM
Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK.
Nyitrai
Miklós
M
Alonso
María C
MC
Weiss
Stefan
S
Geeves
Michael A
MA
Cross
Robert A
RA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019281:Dimerization; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D008970:Molecular Weight; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D010782:Photolysis; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D012996:Solutions
D010710:Phosphates; D012996:Solutions; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins