Crystallization of Kinesin Family Motor Proteins

Crystallization


Motor proteins of several kinesin family groups have now been crystallized:
monomeric Kinesin-1 motor domains from human, rat and Neurospora (Kull et al., 1996; Sack et al., 1997; Song et al., 2001) and
dimeric rat Kinesin-1 (two motor domains connected by a short coiled-coil; Kozielski et al., 1997);

the Kinesin-14 (formerly C-terminal motor) proteins Drosophila Ncd in its monomeric and dimeric forms (Sablin & Fletterick, 1995, Sablin et al., 1996, 1998; Kozielski et al., 1997; Yun et al., 2003), yeast KAR3 (monomer) (Gulick et al., 1998; Yun et al., 2001) and three KAR3 mutants (Yun et al., 2001), and KCBP (Vinogradova et al., 2004);

the Kinesin-3 (formerly Unc104/KIF1) motor, KIF1A bound to different nucleotides (KIF1A-ADP, KIF1A-AMPPCP, KIF1A-ADP-Vi, KIF1A-AlFx) (Kikkawa et al., 2001; Nitta et al., 2004);

the Kinesin-5 (formerly BimC) motor, monomeric Eg5 (Turner et al., 2001);
and the Kinesin-13 (formerly MCAK) motor, PfKinI (Shipley et al., 2004).

The following tables summarize the crystallization conditions and some of the crystal parameters.

Crystals of monomeric rat Kinesin-1



Table 1: Crystallization conditions for kinesin motor protein constructs

Construct Method Conditions References
Human Kinesin-1

hK349
Sitting drop
4°C
5 mg/ml protein in 50 mM Na-acetate, pH 4.6, 75 mM KCl, 3.5% (w/v) PEG 4000, 2.5 mM ATP, 10 mM MgCl2

Reservoir: 100 mM Na-acetate pH 4.6, 150 mM KCl, 7% PEG 4000, 5 mM ATP, 20 mM MgCl2
Kull et al., 1996
Ncd 335-700 Sitting drop
Room temperature
7 mg/ml protein in 10 mM Pipes, pH 7.2, 100 mM NaCl, 1 mM EGTA, 1 mM DTT, 7% (w/v) PEG 4000, 0.3% octyl-ß-D-glucoside, 2 mM ATP, 10 mM MgCl2

Reservoir: 15% (w/v) PEG 4000, 60 mM NaCl equally buffered

Sablin & Fletterick, 1995


Sablin et al., 1996
Ncd 293-700 Hanging drop
18°C
17 mg/ml protein in 20 mM HEPES pH 7.4, 200 mM NaCl, 10 mM MgCl2, 2 mM DTT was pre-incubated with 4 mM AMP*PNP or ATP for 2 hours

Crystals grew in 11% PEG 8000, 0.8 M NaCl, 50 mM Na2H2PO4, pH 6.8, 7 mM DTT
Yun et al., 2003
Rat Kinesin-1

rK354
Hanging drop
Room temperature
9-14 mg/ml protein 20 mM PIPES pH 7.5, 50 mM KCl, 1 mM EGTA, 1 mM DTT, 0.9 M Li2SO4

Reservoir: 20 mM PIPES pH 7.5, 1.8 M Li2SO4
or

15 mg/ml protein in 10 mM PIPES pH 7.5, 50 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.5 mM NaN3, 0.9M (NH4)2SO4

Reservoir: 1.8M (NH4)2SO4,50 mM NaCl
Kozielski et al., 1997a

Sack et al., 1997
Rat Kinesin-1

rK379
Hanging drop
Room temperature
15 mg/ml protein 10 mM PIPES pH 7.5, 200 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.5 mM NaN3, 0.8M (NH4)2SO4

Reservoir: 1.6M (NH4)2SO4, 200mM NaCl
Kozielski et al. 1997a,b
Kar3 383-729 Microbatch
Room temperature
11 mg/ml protein 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM MgCl2, 0.2 mM NaN3, 2 mM ADP combined 1:1 with 22% methyl ether PEG 2000, 100 mM NaCl, 2% ethylene glycol, 50 mM HEPES pH 7.0 Gulick et al., 1998
Ncd 281-700 Sitting drop
4°C
20 mg/ml protein in 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, 0.7 M Li2S04, 2 mM ADP, 10 mM MgCl2

Reservoir: 20 mM HEPES pH 7.5, 1.4 M Li2SO4, 1 mM EGTA, 1 mM DTT, 10 mM MgCl2

Sablin et al., 1998
Ncd 295-700 Hanging drop
Room temperature
5 mg/ml protein in 25 mM Na2PO4, pH 6.8, 6.8% PEG 8000, 1 M NaCl, 2 mM ATP, 3.5 mM DTT, 10 mM MgCl2

Reservoir: 25 mM Na2PO4 pH 6.8, 13.5% PEG 8000, 2 M NaCl, 7 mM DTT
Kozielski et al., 1999
Eg5 1-368

HsKSP
Sitting Drop
4°C
5 mg/ml protein in 9% PEG-3350, 50 mM PIPES pH 6.8, 100 mM NaNO3

Reservoir: 18% PEG-3350, 100 mM PIPES pH 6.8, 200 mM NaNO3

Imperfect crystals were crushed and used to seed 5 mg/ml Eg5 in 7.5% PEG-3350, 50 mM MES pH 5.6, 100 mM NaNO3

Reservoir: 15% PEG-3350, 100 mM MES pH 5.6, 200 mM NaNO3
Turner et al., 2001
KIF1A-ADP Vapor diffusion 2 microliters of protein (15 mg/ml) + 2 microliters of reservoir buffer (RB1) composed of 30% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate, 8% w/v sucrose, 4 mM ADP, 10 mM MgCl2, equilibrated against RB1 for 5 d Kikkawa et al., 2001
KIF1A-AMPPCP
(soaked)
Vapor diffusion 15 mg/ml protein + reservoir buffer (RB2) composed of 27% w/v PEG4000, 100 mM MES-NaOH pH 6.5, 200 mM sodium acetate, then soaked in RB2 + 20 mM AMPPCP + 40 mM MgCl2 for 24 hrs. Kikkawa et al., 2001
KIF1A-AMPPCP
(co-crystalized)
Vapor diffusion 15 mg/ml protein co-crystalized with 5 mM AMPPCP + 20 mM MgCl2 in reservoir buffer (RB3) composed of 30% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate Kikkawa et al., 2001
Nkin 1-355 Sitting Drop
19°C
7.5-15 mg/ml protein in 20 mM Tris pH 7.9, 5 mM MgCl2, 0.5 mM ADP

Reservoir: 17.5% PEGMME 2000, 100 mM HEPES pH 6.5-7.5, 3% glycerol
Song et al., 2001
Kar3 +N11 (WT) Hanging Drop
18°C
2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 Yun et al., 2001
Kar3 N650K Hanging Drop
18°C
2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 Yun et al., 2001
Kar3 R598A Hanging Drop
4°C
2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 Yun et al., 2001
Kar3 E631A Hanging Drop
4°C
2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 Yun et al., 2001
Ncd 293 – 700 18°C 17 mg/ml protein in 20 mM HEPES pH 7.4, 200 mM NaCl, 10 mM MgCl2 and 2 mM DTT, pre-incubated with 4 mM AMP PNP or ATP for 2 h
Crystals grew in 11.0% PEG 8000, 0.8 M NaCl, 50 mM Na2HPO4/NaH2PO4 (pH 6.8) and 7 mM DTT at 18°C
Yun et al., 2003
KIF1A Vapor diffusion
at 20°C for 24 h
AMPPNP: 27% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium
acetate and 3% w/v xylitol with a final concentration of 5 mM AMPPNP and 1 mM MgCl2

ADP-AlFx: 29% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate
and 3% w/v xylitol with a final concentration of 1 mM ADP, 1 mM MgCl2 and 1 mM AlF3

ADP-Vi; 28% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium
acetate and 3% w/v xylitol with a final concentration of 1 mM ADP, 1 mM MgCl2 and 1 mM NaVO4

Nitta et al., 2004
KCBP
884 – 1252
Sitting drop
vapor diffusion
at 4°C
10 mg/mL protein in 50 mM Tris, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 1 mM ATP, 1 mM Tris(2-carboxyethyl)-phosphine
Reservoir: 20% polyethylene glycol 3350 in 0.2 M di-sodium hydrogen phosphate, pH 9.1
Vinogradova et al., 2004
KinI Sitting drop
4°C
10 – 20 mg/ml protein
Reservoir: 1.4-1.8 M ammonium sulfate, 100 mM sodium acetate (pH 5.0), 200 mM sodium nitrate
Shipley et al., 2004


Table 2: Crystallographic parameters


Construct Space group Unit cell Resolution Structural determination Special Features
hK349

PDB: 1BG2
P212121 a=48.54 Å

b=67.94 Å

c=112.95 Å
1.8 Å Multiple isomorphous replacement
rK354

PDB: 2KIN
P212121 a=71.56 Å

b=73.67 Å

c=74.13 Å
1.9 Å Molecular replacement
rK379


PDB: 3KIN
P212121 a=72.2 Å

b=91.9 Å

c=141.7 Å
3.0 Å Multiple isomorphous replacement
Kar3 382-729


PDB: 3KAR
P21 a=44.1 Å

b=81.2 Å

c=48.3 Å

ß=105.8°
2.3 Å Molecular replacement and phases of three heavy atom derivatives
Ncd 335-700

Coordinates
I222 a=127.1 Å

b=122.3 Å

c=68.0 Å
2.5 Å Multiple isomorphous replacement
Ncd 281-700

PDB: 2NCD
P6122 a= 123.0 Å

b=123.0 Å

c=121.1 Å
2.5 Å Molecular replacement
Ncd 295-700


PDB: 1CZ7
C2221 a=116.19 Å

b=148.83 Å

c=261.52 Å
2.9 Å Molecular replacement and phases of three heavy atom derivatives
Eg5

PDB: 1II6
P21 a=53.08 Å

b=78.59 Å

c=94.15 Å

ß=93.84°
2.1 Å Molecular replacement
KIF1A-ADP


PDB: 1I5S
P212121 a=41.67 Å

b=51.92 Å

c=157.06 Å
2.2 Å Molecular replacement
KIF1A-AMPPCP
(soaked)


PDB: 1I6I
P212121 a=41.99 Å

b=56.40 Å

c=156.12 Å
2.0 Å Molecular replacement Motor bound to AMPPCP
KIF1A-AMPPCP
(co-crystalized)


PDB: 1IA0
P212121 a=42.42 Å

b=55.43 Å

c=157.27 Å
1.9 Å Molecular replacement Motor bound to AMPPCP
Nkin 1-355

PDB: 1GOJ
P212121 a=51.97 Å

b=72.73 Å

c=84.93 Å
2.3 Å Molecular replacement
Kar3+N11 (WT)


PDB: 1F9T
P21 a=43.6 Å

b=78.8 Å

c=47.2 Å

ß=105.0°
1.5 Å Molecular replacement
Kar3 N650K


PDB: 1F9U
P21 a=43.6 Å

b=78.0 Å

c=47.3 Å

ß=105.1°
1.7 Å Molecular replacement
Kar3 R598A


PDB: 1F9V
P21 a=43.9 Å

b=77.4 Å

c=47.7 Å

ß=105.9°
1.3 Å Molecular replacement
Kar3 E631A


PDB: 1F9W
P43 a=62.9 Å

c=153.6 Å

2.5 Å Molecular replacement
Ncd 293 – 700


PDB: 1N6M
C2 a= 162.6Å

b= 66.6Å

c= 94.8Å

2.5 Å Molecular replacement Rotation of stalk by 75°
KIF1A

AMPPNP:
PDB: 1VFV
PDB: 1VFW

ADP-AlFx:
PDB: 1VFX

ADP-Vi:
PDB: 1VFZ

AMPPNP1:
P212121

AMPPNP2:
P212121

ADP-AlFx:
P212121

ADP-Vi:
P212121


AMPPNP1:
a= 42.6 Å

b= 55.2 Å

c= 157.0 Å

AMPPNP2:
a= 42.6 Å

b= 55.5 Å

c= 156.7 Å

ADP-AlFx:
a= 41.9 Å

b= 54.6 Å

c= 156.8 Å

ADP-Vi:
a= 41.5 Å

b= 51.8 Å

c= 157.0 Å

AMPPNP1: 1.85 Å

AMPPNP2: 2.2 Å

ADP-AlFx: 2.6 Å

ADP-Vi: 2.2 &#197

Molecular replacement Motor bound to AMPPNP, ADP-AlFx or ADP-Vi
KCBP


PDB: 1SDM
P21212
a= 95.7 Å

b= 85.3 Å

c= 44.5 Å

2.3 Å Molecular replacement
KinI


PDB: 1RY6
P3221
a= 105.6 Å

c= 84.8 Å

1.6 Å Molecular replacement No nucleotide bound to motor

Contributed by J. Muller, J. Kull and E. Mandelkow




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Created 11 October 1999 17:30 GMT

Modified 29 October 2004 11:24 GMT