Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.

ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate by S-Sepharose chromatography, and trace amounts of contaminants were removed by passing through a MonoQ column. The yield was 20 mg from a 500 mL. culture of E. coli. The purified ncd motor domain exhibited an unusual UV spectrum with a broad peak around 272-275 nm, which was at least partly due to the bound nucleotide. Upon incubation with radioactive ATP, 3H at adenine but not 32P at y-phosphate was retained by the protein on gel filtration, indicating it bound ADP but not ATP. Thus, like kinesin, nucleotide binding to the ncd motor domain is tight, although there is an equilibrium between the protein and free nucleotide. We also used a fluorescent ATP analogue, mantATP, for the kinetic study of ncd motor domain. MantATP was turned over by ncd motor domain slowly in the absence of microtubules, but microtubules activated the turnover to a similar extent to that of ATP. Upon incubation with ncd motor domain, the fluorescent intensity of mantATP increased at 0.005 s-l, which is likely to reflect the release of endogenous ADP and incorporation of mantATP into the protein. The fluorescence intensity of the ncd motor domain having bound mantADP, likewise, decreased upon mixing with ATP, representing the mantADP release. The rate was accelerated more than 1000-fold to 3.3 s-l by the presence of saturating microtubules. The profiles of the mantADP release rate and the mantATP turnover rate versus microtubule concentration were nearly identical, except that the maximal rate of mantATP turnover was 30% lower than the maximal mantADP release rate. This result suggests that mantADP release substantially contributes to the overall cycle time. We have also measured the equilibrium dissociation constants for ncd motor domain binding to microtubules in the presence of ADP [&(ADP) = 4-5 pM] and ATP [&(ATP) = 6-7.5 pM] and the apparent halfmaximal microtubule stimulation of ATPase activity (5-7 pM) under an identical condition. The enzymatic and microtubule-binding characteristics of ncd motor domain reported here are similar to those of kinesin. The notable exception, however, is that &(ADP) for kinesin is 2-3-fold larger than &(ATP), which suggests the existence of a weak binding ADP state in the case of kinesin but not in the case of ncd motor domain. Such a difference could be relevant for understanding the opposite polarity of movement of these two microtubule motors.Eukaryotes generate cytoplasmic motility using two types of filaments, actin and microtubules. Actin-based motility is driven by motors that belong to the myosin superfamily

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2-3-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, MtN ؉ T L | ; There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N⅐T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN⅐D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt⅐Ncd complex is not processive.The non-claret disjunctional (Ncd) 1 protein is a member of the sub family of kinesin proteins, which have the motor domain at the C-terminal end of the polypeptide chain and move toward the negative end of the microtubule (1). The velocity of Ncd movement of 100 -200 nm s Ϫ1 (2, 3) is about five times slower than neuronal kinesin. The motor domain of approximately 350 amino acid residues shows 40% homology with kinesin, and the three-dimensional structures of the Ncd and kinesin motor domains are similar (4, 5).Previous kinetics studies (6 -8) have shown that there are similarities in the reaction mechanism despite the difference in rate and polarity of movement. The dissociation of ADP is the rate-limiting step in ATP hydrolysis in the absence of microtubules, and the rate is markedly increased by microtubule binding. The rate of ADP dissociation makes a major contribution to rate-limiting the fully activated ATPase based on measurements of the maximum rate of dissociation of ADP in the reaction of the motor-ADP complex with microtubules. The ratio of the rate of ADP dissociation to V m is approximately 1.5 for Ncd, and a similar ratio was obtained for various kinesin constructs (9, 10). However, Gilbert et al. (11) obtained a much higher rate of dissociation of ADP for a Drosophila kinesin and concluded that ADP dissociation was not rate-limiting.The present work was undertaken to provide a more detailed kinetic scheme for an Ncd monomer and to compare the scheme with a kinesin monomer (human kinesin construct K332). A difficult problem in studying Ncd is that removal of the strongly bound ADP leads to aggregation and denaturation of the protein. The microtubule-Ncd complex is sufficiently stable in the absence of bound nucleotide to permit kinetic measurements to be made for comparison with the microtubule-kinesin complex.The microtubule-activated Ncd is very similar to kinesin in showing a phosphate burst phase, and the hydrolysis of ATP is rate-limited by ADP release. It could be approximately described as a slow kinesin. However, the relative rates of some steps in the mechanism, including the rates of dissociation of the Mt⅐Ncd complex compared with t...

The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209-700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K(d) values are 1.0 microM for ADP-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for ADP, and 10 microM for ADP-vanadate (in 25 mM NaCl, 22 degrees C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 microM(-1) s(-1). Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme,[see text] where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k(1) and k(-4) are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1-mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1.ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.