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How molecular motor acts
2006.11.27. graduate school of medicine, the University of Tokyo Nobutaka Hirokawa
Life Science Seen from Molecular Motor
Low-angle, rotary shadowing electronmicrograph
Kinesin
KIF1A
Okada et al.Science 283:1152-, 1999
Optical Trapping Nanometry
Spatial resolution < 0.2 nmTemporal resolution < 1 ms
0.2μm beads⇒Small viscous drag
⇒ High temporal resolution
(Response time ≦ 0.25 ms)
Okada,Higuchi, and Hirokawa Nature 424:574-2003
Immobilization of KIF1A to Bead
Bidirectional and Step-wise Movement of a Single KIF1A Monomer
One ATP Hydrolysis Triggers One Stepping Movement.
Time (s)8 9 10
4080
120
-20 0 20
(nm)
(ms)-400
4080
120160
Cycle Time (ms)
0
200
400
600
800
0 200 400 600 800
Step size of KIF1A is 8×n nm, and varies stochastically.
02468
10
-32 32160-16step (nm)
12
Plus-end biased binding of KIF1A to the microtubule: ensemble average
Time (s)
on
0
2
4
-0.2 0 0.2
// MT⊥ MTControl
+-end
//^
Flush Ratchet Model
α β α β α βα β
- +
KIF1AATP
Phosphate
drelease ~0 nm
Dwell Detach
Load F Drag -gv
1D-diffusion under load
dbind ~3 nm
ADPBind
① ②
③④
Microtubule Structure Kikkawa et al.
JCB, 1994: Nature, 1995
Microtubule Microtubule-Kinesin complex
KIF1A-Microtubule Complex: cryo EM(15Å resolution)
Kikkawa et al. Cell 100: 241-, 2000
KIF1A-Microtubule Complex: cryo EM(15Å resolution)
ADP ATP(AMPPNP)
View from outside
ADP ATPView from outside
Tarzan Model
“Drag force” = diffusion might reflect the fluctuation of theflexible tether between KIF1A and tubulin (K-loop & E-hook).
TarzanTarzan
k i
Kikkawa et al. Nature 411: 439- ,2001
Structural Biological Analysis of kinesin motors
KIF1A alternately uses two loops to bind microtubules
Nitta,R. et al. Science 305:678- , 2004
Construction of KIF1A Motor Domain
Chemo-mechanical cycle of kinesin motors
Overall architecture of KIF1A
ATP(AMPPNP)
ADP-Pi (1)(ADP-AlF3)
ADP-Pi (2)(ADP-VO4)
ADP
Conformational changes of two switch regions
KIF1A*=ATP- Microtubule
KIF1A=ADP-Pi -Microtubule
KIF1A*=ADP-Pi -Microtubule
KIF1A=ADP -Microtubule
Nucleotide construct
wild type L12 L11 L8
AMPPNP 4.2±1.3 6.0±1.4 20.2±4.0 25.0±6.0
ADP 6.8±2.5 23.5±8.4 12.3±4.0 26.5±5.0
ATP 10.8±1.8 40.5±11.8 nd nd
ADP-AlFx 5.9±1.5 7.1±1.7 nd nd
ADP-Vi 21.4±4.3 167±66 nd nd
Equilibrium Binding Constants of KIF1A mutant
Design principle
of Walker-
type NTPase
s
Design principle of Walker-type NTPases
Model for the processive movement of the monomeric kinesin KIF1A
Univ. Tokyo
Takao NAKATAYoshimitsu KANAIYasuko NODASumio TERADAYosuke TAKEISen TAKEDAYasushi OKADAYosuke TANAKAMasahiko KAWAGISHIMitsutoshi SETOU
Laurent GUILLAUDRichard WONGJunlin TENGHarukata MIKIDae-Hyung SEOGChunjie ZHAOTerunaga NAKAGAWAMasahide KIKKAWAHiroaki YAJIMARyo NITTATadayuki OGAWA
UCSFRobert J. FletterickElena P. Sabrin
Univ. Hospital TokyoJunko TAKITAYasuhide HAYASHI
Niigata Univ. Brain Res. Inst.
Masaaki SAITOShoji TSUJI