Abstract
Motor domains of kinesin were expressed that extend from the N terminus to positions 346, 357, 365, 381, and 405 (designated DKH346-DKH405) to determine if the kinetic differences observed between monomeric DKH340 and dimeric DKH392 (Hackney, D. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6865-6869) were specific to these constructs or due to their oligomeric state. Sedimentation analysis indicated that DKH346, DKH357, and DKH365 are predominantly monomeric and that DKH381 and DKH405 are predominantly dimeric at 0. 01-0.03 microM, the concentrations used for ATPase assays. In buffer with 25 mM KCl, all have high kcat values of 38-96 s-1 at saturating microtubule (MT) levels. Monomeric DKH346, DKH357, and DKH365 have K0.5(MT) values of 17, 9, and 1.4 microM, respectively, but the K0. 5(MT) values for the dimeric species are significantly lower, with 0. 02 and 0.14 microM for DKH381 and DKH405, respectively. The three new monomers release all of their ADP on association with microtubules, whereas the two new dimers retain approximately half of their ADP, consistent with the half-site reactivity observed previously with dimeric DKH392. Both the kbi(ATPase) (=kcat/K0. 5(MT)) values for stimulation of ATPase by MTs and the kbi(ADP) for stimulation of ADP release by MTs were determined in buffer containing 120 mM potassium acetate. The ratio of these rate constants (kbi(ratio) = kbi(ATPase)/kbi(ADP)) is 60-100 for the dimers, indicating hydrolysis of many ATP molecules per productive encounter with a MT as observed previously for DKH392 (Hackney, D. D. (1995) Nature 377, 448-450). For the monomers, kbi(ratio) values of approximately 4 indicate that they also may hydrolyze more than one ATP molecule per encounter with a MT and that the mechanism of hydrolysis is therefore fundamentally different from that of actomyosin. DKH340 is an exception to this pattern and may undergo uncoupled ATP hydrolysis.
Highlights
The N-terminal 340 amino acids of kinesin are conserved among all superfamily members and contain the sites for ATP hydrolysis and interaction with MTs
To better localize the regions required for dimerization and to determine the influence of dimerization itself, kinesin head domains terminating at a series of positions in the neck region were expressed in Escherichia coli and purified essentially as described previously [11, 20]
At a concentration of Յ1 M, DKH346, DKH357, and DKH365 have s20,w values of 3.5–3.6 S, which are similar to the values for DKH340, which was previously shown to be monomeric [11], whereas DKH381 and DKH405 have s20,w values of 5.1–5.7 S, which are similar to the 5.2 S value for dimeric DKH392 [11]
Summary
(Received for publication, July 25, 1996, and in revised form, December 16, 1996). Wei Jiang, Maryanne F. Previous work has established that DKH340 (containing amino acids 1–340) is monomeric, with an s20,w value of 3.3 S, whereas DKH392 (containing amino acids 1–392) is dimeric, with an s20,w value of 5.2 S [11] This led to a revised domain model of kinesin in which the two minimal motor domains are dimerized through interaction of the coiled-coil region in the neck without a highly flexible hinge at their point of attachment to the neck [11]. The work presented here is an examination of seven head constructs of Drosophila kinesin that differ in the amount of the neck domain that is included This extensive series of constructs allows systematic evaluation of the influence of the length of the neck on the oligomeric state as well as evaluation of the influence of dimerization on kinetic properties under uniform assay conditions. Several characteristic differences between dimers and monomers have emerged
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