EXPRESSION, PURIFICATION, ATPASE PROPERTIES, AND MICROTUBULE-BINDING PROPERTIES OF THE NCD MOTOR DOMAIN

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, H-3 at adenine but not P-32 at gamma-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(-1), 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(-1) 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 [K-d(ADP) = 4-5 mu M] and ATP [K-d(ATP) = 6-7.5 mu M] and the apparent half-maximal microtubule stimulation of ATPase activity (5-7 mu M) 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 K-d(ADP) for kinesin is 2-3-fold larger than K-d(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.