Influence of the Kinesin Neck Domain on Dimerization and ATPase Kinetics*

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 μM, 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 μM, respectively, but the K0.5(MT) values for the dimeric species are significantly lower, with 0.02 and 0.14 μM 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 ∼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.

[1]  J. Cronan,et al.  Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface. , 1994, The Journal of biological chemistry.

[2]  R. Stewart,et al.  Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[3]  D. Hackney.  The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule. , 1994, The Journal of biological chemistry.

[4]  E. Taylor,et al.  A kinetic study of the kinesin ATPase. , 1992, The Journal of biological chemistry.

[5]  D. Hackney,et al.  Kinesin ATPase: rate-limiting ADP release. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Ronald D. Vale,et al.  Crystal structure of the kinesin motor domain reveals a structural similarity to myosin , 1996, Nature.

[7]  D. Hackney,et al.  The kinetic cycles of myosin, kinesin, and dynein. , 1996, Annual review of physiology.

[8]  K. Johnson,et al.  Purification and characterization of two monomeric kinesin constructs. , 1996, Biochemistry.

[9]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[10]  Ting Huang,et al.  Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization. , 1994, The Journal of biological chemistry.

[11]  W. Jiang,et al.  Monomeric Kinesin Head Domains Hydrolyze Multiple ATP Molecules before Release from a Microtubule* , 1997, The Journal of Biological Chemistry.

[12]  E. Taylor,et al.  Mechanism of microtubule kinesin ATPase. , 1995, Biochemistry.

[13]  James C. Lee,et al.  [36] Preparation of tubulin from brain , 1982 .

[14]  D. Hackney,et al.  Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Toshio Yanagida,et al.  Direct observation of single kinesin molecules moving along microtubules , 1996, Nature.

[16]  K. Johnson,et al.  Pre-steady-state kinetics of the microtubule-kinesin ATPase. , 1994, Biochemistry.

[17]  A. Hudspeth,et al.  Movement of microtubules by single kinesin molecules , 1989, Nature.

[18]  C. Penney,et al.  A simple microassay for inorganic phosphate, II. , 1978, Analytical biochemistry.

[19]  K. Johnson,et al.  Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli. , 1993, Biochemistry.

[20]  L. Goldstein,et al.  Evidence that the stalk of Drosophila kinesin heavy chain is an alpha- helical coiled coil , 1992, The Journal of cell biology.

[21]  R A Milligan,et al.  Kinesin follows the microtubule's protofilament axis , 1993, The Journal of cell biology.

[22]  E. Taylor,et al.  Kinetic mechanism of kinesin motor domain. , 1995, Biochemistry.

[23]  J. Correia,et al.  Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341. , 1995, Biochemistry.

[24]  E. Taylor,et al.  Energetics and mechanism of actomyosin adenosine triphosphatase. , 1976, Biochemistry.

[25]  G. Bloom,et al.  Motor proteins. 1: kinesins. , 1994, Protein profile.

[26]  L. Goldstein,et al.  Bead movement by single kinesin molecules studied with optical tweezers , 1990, Nature.

[27]  J. Scholey,et al.  Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains , 1988, The Journal of cell biology.

[28]  R. Cross,et al.  ADP release is the rate‐limiting step of the MT activated ATPase of non‐claret disjunctional and kinesin , 1995, FEBS letters.

[29]  R. Cross,et al.  Kinesin and ncd bind through a single head to microtubules and compete for a shared MT binding site. , 1995, Journal of molecular biology.

[30]  J. Gelles,et al.  Failure of a single-headed kinesin to track parallel to microtubule protofilaments , 1995, Nature.

[31]  J. Howard,et al.  The movement of kinesin along microtubules. , 1996, Annual review of physiology.

[32]  D. Hackney,et al.  Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli. , 1994, The Journal of biological chemistry.

[33]  S. Rosenfeld,et al.  Equilibrium Studies of Kinesin-Nucleotide Intermediates (*) , 1996, The Journal of Biological Chemistry.

[34]  D. Hackney,et al.  Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains , 1995, Nature.