Origins of reversed directionality in the ncd molecular motor.

Lockhart, Andrew; Cross, Robert A.

doi:10.1002/j.1460-2075.1994.tb06317.x

Cited by 58 publications

(79 citation statements)

References 23 publications

(23 reference statements)

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“…The amounts as well as the purity of protein that can be obtained are suitable for kinetic and X-ray crystallography studies. The microtubule-stimulated ATPase rates of ncd motor domain are similar to those described previously for larger ncd constructs containing the motor domain as well as the a-helical coiled coil rod domain (Chandra et al, 1993;Lockhart & Cross, 1994). This indicates that this truncated, monomeric form of the ncd motor has properties that are similar overall to the dimeric ncd motor, although subtle differences may exist.…”

Section: Concentration Of Nacl (M)supporting

confidence: 83%

“…The pH profile in the absence of microtubules was somewhat U-shaped, reminiscent of myosin ATPase (Sekine & Kielley, 1964) or 22s dynein ATPase from Tetrahymena cilia (Shimizu & Kimura, I974), but was high at neutral pH in the presence of microtubules ( Figure 2A). The activity increased with increasing NaCl concentration in the assay mixture in the absence of microtubules in agreement with Lockhart and Cross (1994), but the opposite relation was observed in the presence of microtubules ( Figure 2B). The apparent K m for microtubules of microtubulestimulated ATPase of ncd motor domain increased with ionic strength.…”

Section: Expression and Purification Of Ncd Motor Domainsupporting

confidence: 79%

“…The detachment and rebinding of the motor might involve a different intermediate than in the kinesin cycle. This might be of much importance since the direction of motion could be determined by which intermediate dissociates and rebinds to reset the mechanical cycle, even if the identical "power-generating" conformational changes take place in those motor proteins as discussed by Taylor (1993) and Lockhart and Cross (1994).…”

Section: Discussionmentioning

confidence: 99%

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Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

Shimizu¹,

Sablin²,

Vale³

et al. 1995

Biochemistry

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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

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Section: Concentration Of Nacl (M)supporting

confidence: 83%

Section: Expression and Purification Of Ncd Motor Domainsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

Shimizu¹,

Sablin²,

Vale³

et al. 1995

Biochemistry

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“…Biochemical and structural studies have shown that kinesins from various subfamilies function by similar biochemical mechanisms (Hackney, 1988, Lockhart andCross, 1994;Gilbert et al, 1995;Ma and Taylor, 1995a,b;Crevel et al, 1996;Lockhart and Cross, 1996;Kull et al, 1996;Sablin et al, 1996;Kikkawa et al, 2001). However, certain biochemical and biophysical differences, such as the efficiency of force production, have been noted among members of different subfamilies (Vale and Fletterick, 1997).…”

Section: Introductionmentioning

confidence: 99%

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

Matthies

Baskin

Hawley

2001

MBoC

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NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD Ͼ2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NOD DTW and NOD "DR2" ) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport.

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“…Although their directions of movement along microtubules are in opposite directions, the motor domain of ncd is highly homologous to that of kinesin, with 41% amino acid sequence identity and almost identical structural folds Sablin et al, 1996). Also, it has been shown that the kinetics of the ATPase of ncd is similar to that of kinesin, especially in that ADP release is the rate-limiting step and that microtubules greatly accelerate this step (Hackney, 1988(Hackney, , 1994Sadhu and Taylor, 1992;Lockhart and Cross, 1994;Lockhart et al, 1995a;Shimizu et al, 1995;Crevel et al, 1996;Pechatnikova and Taylor, 1997).…”

Section: Introductionmentioning

confidence: 99%

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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Origins of reversed directionality in the ncd molecular motor.

Cited by 58 publications

References 23 publications

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain

Orphan Kinesin NOD Lacks Motile Properties But Does Possess a Microtubule-stimulated ATPase Activity

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

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