How kinesin waits between steps.
Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads'), a process driven by a conformational change in the neck-linker domain of kinesin. However, the 'waiting conformation' of kinesin between steps remains controversial-some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.
Mori
Teppei
T
Department of Applied Physics, The University of Tokyo, Tokyo 113-8656, Japan.
Vale
Ronald D
RD
Tomishige
Michio
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006868:Hydrolysis; D007536:Isomerism; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D009068:Movement; D011487:Protein Conformation
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins