ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement. Cell 136 , 110-122 (2009). Cochran JC Kull FJ. Conventional kinesin. Kinesin walking model. Klumpp LM, 2004. Vale RD, 2003. Kinesin family proteins.
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NOD Allows Microtubule End Tracking
and Drives Chromosome Movement
Cell136, 110-122 (2009)
Cochran JC Kull FJ
Kinesin walking model
Klumpp LM, 2004
Vale RD, 2003
Kinesin-7(formerly CENP-E) group and MCAK, a member of theKinesin-13 group have both been shown to be kinetochore kinesin proteins, but localize to different regions of the kinetochore.
Kinesin-4 motors, Xklp1, is required for maintenance of spindle bipolarity and congression of chromosomes to the metaphase plate. Nod, a meiotic kinesin 10 in Drosophila , has been postulated to perform an analogous role in oocyte meiosis of positioning chromosomes on the metaphase plate.
nodistributive disjunction gene, NOD
Distributive disjunction, is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined (achiasmata).
During prometaphase nod protein is localized on oocyte chromosomes
and is not restricted to either specific chromosomal regions or to the kinetochore.
anti-histone antibody (green)
anti-tubulin antibody (red)
HMGN domain, consists of three tandemly repeated
high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos.
HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain.
Structure of Nod
The HMGN and HhH(2)/NDD domains are involved in binding Nod to chromosomes
Cui and Hawley, 2005
NOD has a strong preference for binding to the plus end of MT
NOD, push chromosomes toward the metaphase plate during female meiosis.
the ratio of plus-end (52) to minus end (3) binding events was 17:1
Cui et al., 2005
Fully closed conformation of Sw2
SW2 stabilized by a salt bridge
ATP hydrolysis competent
P101 and P102 mimics the monastrol binding site
Will there be similar kinetic profile of NOD for MT binding and ADP product release?
Nucleotide dependent isomerization is correlated with the “closing” of loop L5, which promotes both tight nucleotide and tight monastrol binding inhibiting the conformational change required for ADP product release.
Cochran and Gilbert, 2005
Ogawa et al., 2004; Vale and Milligan, 2000
NOD’s MT binding region
L8 in NOD contains many hydrophobic residues (M159-A164; sequence MPMVAA) that would potentially contribute to interactions at the MT interface.
Klumpp et al., 2004
(2) Sw2(L11) to α4, L12 and α5
Alternate conformation in NOD’s MT binding region
Alpha 4 clockwise rotation away from alpha-6 could alter the MT interface leading to distinct NOD binding to the MT lattice
Typically, kinesins bind to MTs with high affinity in the presence of nonhydrolyzable ATP analogs such as AMPPNP.
the clockwise rotation of nucleotidefree
NOD relative to nucleotide-free kinesin-1 on the MT is in the opposite direction from the counterclockwise rotation reported
upon binding of ATP analogs to KIF1A or kinesin-1
Ki,AMPPNP=25.7 ± 2.4uM
ADP binds tighter to NOD than ATP
Ki,ADP=1.7 ± 0.2uM
Where NOD‧ADP (1:1) exists in equilibrium between nucleotide-free (～53%) and ADP bound (～ 47%)
MT‧NOD + ADP
Less than 3 fold activation of ADP release which is similar to the kinetics of ADP release for kinesin 5 in the presence of monastrol.
Slight hyperbolic fit
Two step mechanism with a rapid conformational change that tightens substrate binding to NOD
Rapid nucleotide binding promotes dissociation of the MT‧NOD complex
Nucleotide free MT‧NOD complex was rapidly mixed with different nucleotides
Acid quench MT‧NOD complex
ATP hydrolysis kinetics
ATP hydrolysis is rate limiting step.
Typically, ADP product release is the rate limiting step.
A conformational change in Sw1 (L5- α 3) occurs to reach the fully cosed lconformation in establishing the “hydrolysis competent” state.
Pi product release MT‧NOD complex
Slow linear phase in Pi release kinetics even at high ATP conc.
Maximum Pi release rate at 0.016 s-1
A slow reaction occurs before Pi release limits the observed kinetics.
An in vitro MT ‧ NOD ATPase model MT‧NOD complex
Nonmotile kinesin tracks MT plus ends and harnesses the force of MT polymerization to drive the movement of chromosome arms .