Influence of the Kinesin Neck Domain on Dimerization and ATPase Kinetics

Jiang, Wei; Stock, Maryanne F.; Li, Xun; Hackney, David D.

doi:10.1074/jbc.272.12.7626

Cited by 84 publications

(103 citation statements)

References 34 publications

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“…It seems likely that the asymmetric interaction between the two heads of kinesin could be strong and specific enough to be preserved under both crystallizing and EM specimen-preparation conditions. We suggest that this structure is probably close to a conformation that occurs during the hydrolysis cycle and that the asymmetry reflects the known functional asymmetry of dimeric kinesin (Hackney, 1994;Gilbert et al, 1995;Jiang et al, 1997). …”

Section: Alternative Conformations Of Dimeric Kinesin⅐adpmentioning

confidence: 71%

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

Hirose

Löwe

Alonso

et al. 1999

MBoC

100

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We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin⅐ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd)⅐ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd⅐ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules.

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Section: Alternative Conformations Of Dimeric Kinesin⅐adpmentioning

confidence: 71%

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

Hirose

Löwe

Alonso

et al. 1999

MBoC

100

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“…DKH346 and DKH405 are monomer and dimer motor domains of conventional Drosophila kinesin-1 (8). BKinM is DKH346 fused at the N terminus to the N-terminal extension of BimC (9), a member of the kinesin-5 family.…”

Section: Methodsmentioning

confidence: 99%

The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

Hackney

2005

Proc. Natl. Acad. Sci. U.S.A.

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Although the steps for the forward reaction of ATP hydrolysis by the motor protein kinesin have been studied extensively, the rates for the reverse reactions and thus the energy changes at each step are not as well defined. Oxygen isotopic exchange between water and P i was used to evaluate the reverse rates. The fraction of the kinesin⅐ADP⅐P i complex that reverts to ATP before release of Pi during net hydrolysis was Ϸ0 and Ϸ2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P i and the MT⅐kinesin⅐ADP complex was Ϸ1.7 M ؊1 ⅐s ؊1 (K0.5 ADP ‫؍‬ 70 M) with monomeric kinesin in the absence of net hydrolysis. Synthesis of bound ATP from the ADP of the tethered head of a dimer-MT complex was 20-fold faster than for the monomer-MT complex. This MT-activated ATP synthesis at the tethered head is in marked contrast to the lack of MT stimulation of ADP release from the same site. The more rapid ATP synthesis with dimers suggests that the tethered head binds behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the MT and would thus facilitate its docking on synthesis of ATP. The observed rate of ATP synthesis also puts limits on the overall energetics that suggest that a significant fraction of the free energy of ATP hydrolysis is available to drive the docking of the neck linker on binding of ATP.ATPase ͉ energy coupling ͉ motility ͉ motor protein ͉ isotopic exchange K inesin-1 is molecular motor that moves along microtubules (MTs) in a highly processive manner. Scheme 1 presents a minimal mechanism for ATP hydrolysis by the MT complex of a kinesin monomer motor domain (see refs. 1-3 for recent reviews of the ATPase mechanism and coupling to motility). ATP binds rapidly and is hydrolyzed at 100-300 s Ϫ1 . P i release occurs without a lag after hydrolysis, and thus P i release is at least as fast as hydrolysis (k 3 Ն k 2 ). ADP release is partially ratelimiting for conventional kinesin-1 and may be the principal rate limiting step for some other kinesin superfamily members.Another important aspect, however, is the equilibrium constant of each step, because it determines the free energy that is available at that step for coupling to movement against a load. The equilibrium constants can be determined if the rates in the reverse direction are known, but this is difficult because the overall equilibrium strongly favors hydrolysis. Oxygen isotopic exchange is a powerful technique for evaluation of the reversibility of the hydrolysis reaction and has provided important insight into the mechanism of the F1-ATPase (4) and myosin (5, 6). It is used here to determine the rates of the reverse reactions of kinesin.Hydrolysis of ATP by kinesin proceeds through attack of water on the ␥ phosphoryl to yield P i that contains one water-derived oxygen and three oxygens from the nonbridge ␥ oxygens of the ATP, as indicated in Scheme 2 for hydrolysis of unenriched ATP in 100% 18 O-enriched water. If P i release vi...

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“…All reactions were performed at 25°C in A25 buffer as described previously (3). Centrifugation on sucrose density gradients and gel filtration on Bio-Gel A-5m were performed using standard proteins as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

“…The first coiled-coil region that extends from the motor domain is designated the neck region. Constructs that contain the head and the COOHterminal part of the coiled-coil neck form dimers (3). There is a likely hinge at position ϳ400 that marks the boundary between the neck and the stalk.…”

mentioning

confidence: 99%

Formation of the Compact Confomer of Kinesin Requires a COOH-terminal Heavy Chain Domain and Inhibits Microtubule-stimulated ATPase Activity

Stock

Guerrero²,

Cobb

et al. 1999

Journal of Biological Chemistry

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146

145

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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 ϳ6.9 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 ϳ50 amino acids are required for the formation of the compact conformation. Separately expressed COOH-terminal tail segments and NH 2 -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 k cat at saturating microtubules).Kinesin is an ATP-dependent motor protein that is involved in movement of membranous vesicles along MTs 1 (see Refs. 1 and 2). The NH 2 -terminal ϳ340 amino acids of the heavy chain forms a globular motor domain (head) that has MT-stimulated ATPase activity (see Fig. 2B). The motor domain is followed by a long central coiled-coil stalk region and a small nonhelical domain at the COOH-terminal. The central region contains several positions at which the coiled-coil propensity is low and these likely represent hinges in the stalk. The first coiled-coil region that extends from the motor domain is designated the neck region. Constructs that contain the head and the COOHterminal part of the coiled-coil neck form dimers (3). There is a likely hinge at position ϳ400 that marks the boundary between the neck and the stalk. Peptides from the neck (4, 5) and stalk (6) have been demonstrated to interact in a coiled-coil manner by several criteria. The crystal structure of a dimeric head plus neck construct has been recently determined (7) and it directly demonstrates the coiled-coil interactions in the neck region.Native kinesin is a heterotetramer composed of a dimeric heavy chain core with two light chains attached in the COOHterminal region (8, 9). At high salt kinesin exist in an extended conformation with an s 20,w value of ϳ6 S, but adopts a more compact conformation at low salt concentration with an s 20,w value of ϳ9 S. This global conformational transition is readily reversible and can be observed b...

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Influence of the Kinesin Neck Domain on Dimerization and ATPase Kinetics

Cited by 84 publications

References 34 publications

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP

Formation of the Compact Confomer of Kinesin Requires a COOH-terminal Heavy Chain Domain and Inhibits Microtubule-stimulated ATPase Activity

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