ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking

1. ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement Cell136, 110-122 (2009) Cochran JC Kull FJ

2. Conventional kinesin Kinesin walking model Klumpp LM, 2004 Vale RD, 2003

3. 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.

4. 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

5. 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

6. 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)

7. 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?

8. 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

9. 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

10. 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

11. (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

12. 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

13. 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

14. Kinetics of MT‧NOD complex formation Stopped flow Fast/slow phase

15. 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%)

16. 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?

17. 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

18. 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

19. 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.

20. 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.

21. An in vitro MT ‧ NOD ATPase model

22. 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