Kinetic evidence for low chemical processivity in ncd and Eg5.
The kinesin molecular motor "walks" processively along microtubules, touching down with alternate motor domains and transiently bridging between sites spaced 8 nm apart axially. To allow bridging, the coiled coil tail of kinesin would need to unzip a region immediately adjacent to the heads, and the tail region sequence at this point indeed contains potentially destabilising interruptions in the regular hydrophobic heptad repeat. We noticed that such interruptions are substantially absent from the coiled coil tails of Eg5, a slow kinesin homologue, and ncd, a reverse-directed homologue, and we wondered if this precluded their processivity. We measured the temperature dependence of kcat/K50% MTs, an index of the chemical processivity of a motor, the number of ATPs split per productive diffusional encounter of motor with microtubule. We found two-headed ncd (GSTMC5) and two-headed Eg5 (E437GST) constructs to be slightly if at all processive in solution over the range 4 degrees C to 30 degrees C. By contrast, two-headed kinesin constructs K401 and K430 were processive, and became substantially more so with increasing temperature. Arrhenius plots for the solution ATPase were linear for all three motors. Arrhenius plots for MT gliding assays were linear and essentially parallel for E437GST and GSTMC5 (Ea = 61 and 63 kJ mol-1) but for K430 the plot was biphasic, with a break at 17 degrees C, corresponding to a 30% reduction in Ea from 84 to 57 kJ mol-1. The data indicate that ncd and Eg5 are only slightly if at all processive, and suggest that this may be related to structural differences in their coiled coil neck regions.
Crevel
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Molecular Motors Group, Marie Curie Research Institute, Oxted, Surrey, UK.
Lockhart
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Cross
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D029721:Drosophila Proteins; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism; D029867:Xenopus Proteins
D029721:Drosophila Proteins; C088335:KIF11 protein, Xenopus; D014404:Tubulin; D029867:Xenopus Proteins; C066696:ncd protein, Drosophila; D000251:Adenosine Triphosphatases; D016547:Kinesins