graduate school of medicine, the University of Tokyo

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