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D D Hackney

Publications and source records attributed to D D Hackney.

At least 19 recordsLinked to original sources
PubMed2000

Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release.

Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883-937 of the kinesin alpha-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.

Adenosine Diphosphate↗
PubMed1999

Formation of the compact confomer of kinesin requires a COOH-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity.

Full-length Drosophila kinesin heavy chain from position 1 to 975 was expressed in Escherichia coil (DKH975) and is a dimer. The sedimentation coefficient of DKH975 shifts from 5.4 S at 1 M NaCl to approximately 6.9 S at <0.2 M NaCl. This transition of DKH975 between extended and compact conformations is essentially identical to that for the heavy chain dimer of bovine kinesin (Hackney, D. D., Levitt, J. D., and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701). Thus the capacity for undergoing the 7 S/5 S transition is an intrinsic property of the heavy chains and requires neither light chains nor eukaryotic post-translational modification. DKH960 undergoes a similar transition, indicating that the extreme COOH-terminal region is not required. More extensive deletions from the COOH-terminal (DKH945 and DKH937) result in a shift in the midpoint for the transition to lower salt concentrations. DKH927 and shorter constructs remaining extended even in the absence of added salt. Thus the COOH-terminal approximately 50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH2-terminal head/neck segments interact in a salt-dependent manner that is consistent with the compact conformer being produced by the interaction of domains from these regions of the heavy chain dimer. The microtubule-stimulated ATPase rate of DKH975 in the compact conformer is strongly inhibited compared with the rate of extended DKH894 (4 s-1 and 35 s-1, respectively, for kcat at saturating microtubules).

Adenosine Triphosphatases↗
PubMed1998

Interaction of mant-adenosine nucleotides and magnesium with kinesin.

Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.

Adenosine Diphosphate↗
PubMed1997

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 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.

Adenosine Diphosphate↗
PubMed1997

Monomeric kinesin head domains hydrolyze multiple ATP molecules before release from a microtubule.

Transient kinetic analysis of microtubule-stimulated ATP hydrolysis by the monomeric kinesin motor domain DKH357 was performed to investigate the kinetic pattern of a monomer. Both ATP and ADP produced dissociation of the complex, microtubule (MT).E, of microtubules with DKH357 at a maximum rate of approximately 45 s-1 as determined by decrease in turbidity. The maximum dissociation rate was independent of the KCl concentration between 25 and 200 mM. At subsaturating levels of nucleotide, ATP was more effective than ADP in dissociating DKH357 from MT.E (1.6 and 0.4 microM-1 s-1 for ATP and ADP, respectively, at 50 mM KCl). Addition of ATP to MT.E results in a burst of product formation with a maximum initial rate of approximately 100 s-1 at saturating levels of ATP. This maximum hydrolysis rate of 100 s-1 is similar to the maximum steady state ATPase rate at saturating microtubules of approximately 70 s-1, and thus hydrolysis is at least partially rate-limiting. When the MT lattice was highly occupied with bound DKH357, the amplitude of the burst was approximately 2 per DKH357 active site (superstoichiometric). The rate constant for the burst transient was approximately 45 s-1, which is the same as the rate for dissociation of DKH357 from the microtubule and this suggests that dissociation and termination of the burst phase are coupled. The size of the burst increased with decreasing initial occupancy of the MT lattice with bound DKH357 and approached the value of approximately 4 ATP molecules predicted by previous steady state measurements (Jiang, W., Stock, M., Li, X., and Hackney, D. D., submitted for publication).

Adenosine Diphosphate↗
PubMed1996

Myosin and kinesin: mother and child reunited.

The recent solution of the crystal structure of the kinesin motor domain reveals striking similarities to the core region of the myosin motor domain, implying a strong evolutionary relationship between these two motors. However, a complete understanding of the way that motility is generated will require additional structural information, which may explain how the two motors have adapted to their fundamentally different linear substrates, F-actin and microtubules.

Animals↗
PubMed1996

The kinetic cycles of myosin, kinesin, and dynein.

The ATPase cycles of the molecular motors myosin, kinesin, and dynein are reviewed, with emphasis on their similarities and differences. Myosin generates motility along actin filaments and functions in muscle contraction, organelle movement, and cytokinesis. Dynein and kinesin produce movement along microtubules. All the motors exhibit burst kinetics with rate-limiting product release. Binding of the products-complex to the filament accelerates product release and completes the ATPase cycle. Kinesin is able to generate processive movement, and the possibilities for how this could be generated by coupling to ATP hydrolysis are discussed.

Adenosine Triphosphatases↗
PubMed1995

Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains.

Studies of immobilized kinesin have shown that a single dimeric molecule can maintain contact with and drive sliding of a microtubule. In solution, however, native kinesin binds microtubules too weakly and hydrolyses ATP too slowly to produce the high sliding velocities seen in motility assay. This apparent inhibition in solution appears to be caused by the binding of kinesin's tail domains to its motor (head) domains in a folded conformation. DKH392, a construct containing two heads but no tails, has been shown to display both tight binding to microtubules and high ATPase rates. Furthermore, it retains one molecule of ADP per dimer when bound to microtubules, which could facilitate a 'hand-over-hand' mechanism for processive motion. Here we show that DKH392 hydrolyses more than 100 ATP molecules per diffusional encounter with a microtubule, even in the high-salt conditions encountered physiologically. This provides direct evidence that kinesin's activity is highly processive, with the motor remaining attached to a microtubule through many cycles of ATP hydrolysis.

Adenosine Diphosphate↗
PubMed1995

Implications of diffusion-controlled limit for processivity of dimeric kinesin head domains.

The diffusion-limited rate for association of the ADP complex of dimeric DKH392 kinesin head domains with a microtubule was estimated to be 2-3 x 10(7) M-1 s-1 based on approximation of a microtubule as a highly elongated prolate ellipsoidal adsorber of 100% efficiency. This theoretical bimolecular rate is approximately 100-fold smaller than the experimental rate, kcat/KMT0.5, for DKH392 that was determined from the stimulation of the steady-state ATPase rate by microtubules. The large difference between these two estimates of the bimolecular rate indicates that it is likely that dimeric DKH392 hydrolyzes multiple ATP molecules during each diffusional encounter with a microtubule.

Adenosine Diphosphate↗
PubMed1994

Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.

The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a dimer in solution that releases only one of its two tightly bound ADP molecules on association with a microtubule, whereas a shorter monomeric construct (designated DKH340) releases > or = 95% of its one bound ADP on association with a microtubule. This half-site reactivity of dimeric DKH392 is observed over a wide range of ratios of DKH392 to microtubules and steady-state ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortuitous balance of rate constants. When [alpha-32P]ATP is included in the medium, incorporation of 32P label into the pool of ADP that is bound to the complex of DKH392 and microtubules occurs rapidly enough for the bound ADP to be an intermediate on the main pathway of ATP hydrolysis. These and other results are consistent with the half-site reactivity being a consequence of the tethering of dimeric DKH392 to the microtubule through one head domain, which is attached in a rigor-like manner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An intermediate of this nature and the tight binding of DKH392 to microtubules in the presence of ATP suggest a mechanism for directed motility in which the head domains of dimeric DKH392 alternate in a sequential manner.

Adenosine Triphosphate↗
PubMed1994

Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.

A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.

Adenosine Diphosphate↗
PubMed1994

Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.

A truncated domain of the alpha-subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH392) extends to amino acid 392 and contains the complete N-terminal region of kinesin which is highly conserved between species. The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues which has been previously characterized as DKH340 (Huang, T.-G., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16493-16501). The s20,w values for DKH340 and DKH392 are 3.3 and 5.2 S and the D20,w values are 7.7 x 10(-7) and 4.9 x 10(-7) cm3 s-1, respectively. These results indicate that DKH340 is a monomer in solution, but DKH392 is a dimer. In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer in contrast to the tight binding of one DKH340 per tubulin heterodimer. Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm.

Adenylyl Imidodiphosphate↗
PubMed1994

The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule.

DKH392 is a construct which contains the first 392 amino acids of the alpha-subunit of Drosophila kinesin and is dimeric in solution (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). The ATPase rate of DKH392 was 0.005 s-1 in the absence of MTs. One ADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis. Microtubules accelerated the rate of ADP release and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (kcat = approximately 45 s-1). The KMT0.5,ATPase value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lower concentrations of DKH392. Physical binding of DKH392 to microtubules in the presence of 1 mM MgATP paralleled saturation of the stimulation of the ATPase activity by microtubules indicating that the rate-limiting step in microtubule-stimulated ATP hydrolysis occurs while DKH392 is bound to the microtubule. These results suggest that microtubule-stimulated ATP hydrolysis by DKH392 may be processive with the hydrolysis of multiple ATP molecules during each diffusional encounter of DKH392 with a microtubule.

Adenosine Diphosphate↗
PubMed1992

Kinesin and myosin ATPases: variations on a theme.

The enzymes kinesin and myosin are examples of molecular motors which couple ATP hydrolysis to directed movement of biological structures. Myosin has been extensively studied and its structure and mechanism of coupling are known in detail. Much less is known about kinesin, but many of its major properties are similar to those of myosin. Both enzymes have two catalytic head groups at the end of a long alpha-helical rod. The head groups contain the sites for ATP hydrolysis and interaction with their respective partners for movement (microtubules or F-actin). In each case the binding and hydrolysis of ATP is rapid and the steady state ATPase rate is limited by a slow step in the region of product release. This slow release of product is accelerated by interaction with actin or microtubules coupled to changes in binding affinity. As there is no evidence for a close evolutionary link between kinesin and myosin, these and other similarities may represent convergence to set of common functional properties which are constrained by the requirements of protein structure and the use of ATP hydrolysis as a source of energy. It will be of particular interest to determine if these common properties are also shared by the large number of divergent proteins which have recently been discovered to possess a domain which is homologous to the head group of kinesin.

Adenosine Triphosphate↗