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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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slide1

ATPase Cycle of the Nonmotile Kinesin

NOD Allows Microtubule End Tracking

and Drives Chromosome Movement

Cell136, 110-122 (2009)

Cochran JC Kull FJ

slide2

Conventional kinesin

Kinesin walking model

Klumpp LM, 2004

Vale RD, 2003

slide3

Kinesin family proteins

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.

slide4

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).

Cheslock, 2005

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)

Afshar,1995

slide5

NOD, a chromokinesin-like protein

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

slide6

Nucleotide sensitive relay

Fully closed conformation of Sw2

Open conformation

1

2

1

2

3

3

SW2 stabilized by a salt bridge

ATPase cycle

Open(ADP)AMPPNPclose

ATP hydrolysis competent

pi+open(ADP)

slide7

Meaning of monastrol binding site?? (cochran and yan)

UNIQUE

P101 and P102 mimics the monastrol binding site

Will there be similar kinetic profile of NOD for MT binding and ADP product release?

slide8

Monastrol affects the ATPae cycle

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

slide9

Communication from the active site to the MT binding region

  • Sw1(L9) to α3 to β5-L8 lobe
  • Sw2(L11) to α4, L12 and α5
  • Changes in MT binding affinity and stabilization of the neck liner in
  • alternate conformation.

Ogawa et al., 2004; Vale and Milligan, 2000

slide10

Sw1(L9) to α3 to β5-L8 lobe

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

slide11

(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

slide12

The affinity of NODs for MTs

NOD Kd,MT=0.072uM

NOD‧ADP Kd,MT=0.69uM

NOD‧ATP Kd,MT=8.0uM

NOD‧AMPPNP Kd,MT=9.4uM

Typically, kinesins bind to MTs with high affinity in the presence of nonhydrolyzable ATP analogs such as AMPPNP.

UNIQUE

slide13

Atypical MT‧NOD binding orientation

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

slide14

Kinetics of MT‧NOD complex formation

Stopped flow

Fast/slow phase

slide15

Competitive inhibition of mantATP binding

Ki,AMPPNP=25.7 ± 2.4uM

ADP binds tighter to NOD than ATP

1

Ki,ADP=1.7 ± 0.2uM

Where NOD‧ADP (1:1) exists in equilibrium between nucleotide-free (~53%) and ADP bound (~ 47%)

slide16

MantADP release kinetics

2

1

MT‧NOD (weak)

MT‧NOD (tight)

MT‧NOD + ADP

isomerization

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.

Meaning?

slide17

MantATP binding kinetics

750 s-1

Slight hyperbolic fit

5.1uM-1s-1

Two step mechanism with a rapid conformational change that tightens substrate binding to NOD

slide18

Rapid nucleotide binding promotes dissociation of the MT‧NOD complex

Nucleotide free MT‧NOD complex was rapidly mixed with different nucleotides

  • There are no apparent lag in the kinetics of dissociation between AMPPNP and ATP.
  • ATP hydrolysis occurs while NOD is off the filament.
  • All other kinesin remain bound to the MT until ATP hydrolyzed

UNIQUE

slide19

Acid quench

ATP hydrolysis kinetics

  • Steady state ATP turnover at 0.016 s-1/site
  • No exponential burst of product formation

ATP hydrolysis is rate limiting step.

Typically, ADP product release is the rate limiting step.

UNIQUE

A conformational change in Sw1 (L5- α 3) occurs to reach the fully cosed lconformation in establishing the “hydrolysis competent” state.

slide20

Pi product release

MDCC-PBP

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.

slide22

Nonmotile kinesin tracks MT plus ends and harnesses the force of MT polymerization to drive the movement of chromosome arms .

Summary

  • NOD binds tightly to MTs in the nucleotide free state
  • Rapid substrate binding leads to NOD detachment from the MT prior to ATP hydrolysis
  • ATP hydrolysis is the rate-limiting step
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