Transcript protws4 4756

CONFORMATIONAL CHANGES AT THE
NUCLEOTIDE-BINDING SITE OF
KINESIN-FAMILY MOTORS
ED PATE
WSU
FORCE GENERATION IN MUSCLE IS
PRODUCED BY THE INTERACTION OF TWO PROTEINS:
ACTIN AND MYOSIN
MYOSIN
ACTIN
MYOSIN
KINESIN
NECK
3-HELIX
CYS-188
CONVERTER
DOMAIN
TRP-510
LOOP 9
SULFHYDRAL
HELIX
NUCLEOTIDE
SWITCH 2
HELIX
SWITCH 1
a3a
NUCLEOTIDE
SWITCH 2
P-LOOP
SWITCH 1
SWITCH 2
P-LOOP
ACTIN BINDING
SITE
NUCLEOTIDE BINDING SITE
MYOSIN
KINESIN
NUCLEOTIDE
NUCLEOTIDE
P-LOOP
P-LOOP
SWITCH 2
SWITCH 1
SWITCH 2
SWITCH 1
COMPARISON OF NCD AND MYOSIN BINDING SITES
P-LOOP
P-LOOP
SWITCH 2
SWITCH 2
NUCLEOTIDE
NUCLEOTIDE
SWITCH 1
SWITCH 1
MYOSIN
NCD
LOCATION OFKINESIN CYS-188
3-HELIX
CYS-188
PROTEIN
S
LOOP 9
O
N
H3 C
H3 C
O
CH3
N
CH3
a3a
NUCLEOTIDE
SWITCH 2
O
P-LOOP
VAN’T HOFF ANALYSIS SHOWS
DH0 = -100 KJ/MOL FOR THE TRANSITION
0.015
Mobile Component (Dashed Line)
Immobile Component (Solid Line)
30° C
Spectrum to Fit (Dashed Line)
Fitted Spectrum (Solid Line)
-R ln[K] (kJ/mol K)
0.01
0.005
0
15° C
-0.005
-0.01
5° C
-0.015
Difference Spectrum
0.0033
0.0034
0.0035
1/T (K)
0.0036
P2
NO2
O
N
N
1'
N
2' O
N
H
P
O
O
O
P
O
P
P4
A
O
O
P3
O
SL-NANTP
O
P1
HN
C
H3 C
ncd +
SSLNANDP
O
CH3
N
H3 C
CH3
O
P5
NO2
H
N
N
N
O
N
C
O
P
O
O
O
O
P
O
O
O
P
O
SSL-NANTP
O
O
H3 C
P2
CH3
N
H3 C
CH3
P4
B
O
NH 2
P3
N
N
O
N
N
5'
O
O
P
O
O
O
2'
O
O
P
O
2',3'-SLATP
O
P1
kinesin +
2',3'SLADP
3'
O
O
C
H 3C
H 3C
P
O
O
CH 3
N
O
CH3
R
2' = O-R 3' = H
2'-SLATP
2' = H 3' = O-R
3'-SLATP
P5
SUMMARY OF CONE ANGLE CHANGES ON
BINDING TO MICROTUBULES
ANALOG
D ANGLE
EPR
ncd
SSL-NANDP
44°
SL-NANDP
>28°
2'-SLADP
12°
3'-SLADP
>12°
2', 3'-SLADP
>16°
kinesin
SSL-NANDP
35°
SL-NANDP
32°
2'-SLADP
>10°
3'-SLADP
11°
2', 3'-SLADP
>18°
CONSTRUCTION OF NCD
WITH SWITCH 1 CLOSED
ADP
P-LOOP
NCD
SWITCH 1
SWITCH 2
CONSTRUCTION OF NCD
WITH SWITCH 1 CLOSED
MYOSIN
SWITCH 1
P-LOOP
NCD
SWITCH 1
SWITCH 2
CONSTRUCTION OF NCD
WITH SWITCH 1 CLOSED
MODIFIED NCD
SWITCH 1
ADP
P-LOOP
NCD
SWITCH 1
12 Å
SWITCH 2
POTENTIAL ENERGY COMPONENTS FROM
BONDED INTERACTIONS
Stretch
H
Flex
O
O
H
H
1
k r (r  req )2

2 BO NDS
H
1
2
k (   eq )

2 angles
H
H
C
H
Torsion
C
H
1
k [1 cos(n   )]

2 dihedrals
O
+
O
qi q j

40 i j rij
1
Coulomb
Interaction
POTENTIAL ENERGY
POTENTIAL ENERGY COMPONENTS FROM
NON-BONDED INTERACTIONS
12
1/r
Pauli Exclusion
Principle
r
6
-1/r
Van der Waals
Force
Aij Bij
 r 12  r 6
i j ij
ij
EQUATIONS OF MOTION FOR
ATOMS
q(t) and qÝ(t) are the position and velocity of an atom
F  ma 
d qÝ  1 [V(q)], V = Potential Energy.
dt
m
Example: Linear Spring.
F  kx; Potential Energy = 1 kx2
2
THE OPEN SWITCH 1 CONFORMATION IS STABLE
ADP
SWITCH 1
P-LOOP
P-LOOP
SWITCH 1
SWITCH 2
THE CLOSED SWITCH 1 CONFORMATION IS STABLE
SWITCH 1
SWITCH 1
P-LOOP
P-LOOP
SWITCH 2
MOLECULAR DYNAMICS RESULTS
• Open structure (x-ray) stable at 300 K
(ADP, ATP, APO)
• Closed structure (model) stable at 300 K
Open and closed structures
Are local minima on a global
free energy surface.
Test with high temperature
Dynamics.
(ADP, ATP, APO)
Energy
barrier
DG
Closed
Open
INFLUENCE OF SWITCH 1 ON EPR PROBES
B
A
Switch 1
open
Switch 1
closed
Adenine
Metal
-phosphate
Nitroxide
D
C
Switch 1
closed
Switch 1
closed
Nitroxide
Nitroxide
Azido group
Azido group
ANGULAR DISTRIBUTION FOR SSL-NANDP IN
THE OPEN AND CLOSED CONFORMATIONS
-90
0
ANGLE
90
SUMMARY OF CONE ANGLE CHANGES
Analog
Cone Angle
Cone Angle
D Angle
D Angle
-MT
+MT
EPR
Model
[100°]
40° [ 50°]
44°
50°
[90°]
44° [ 50°]
>28°
40°
NCD
SSL-NANDP
84°
SL-NANDP
>72°
2´-SLADP
98°
[100°]
86° [ 90°]
12°
10°
3´-SLADP
>134° [110°]
122° [90°]
>12°
20°
ATP HYDROLYSIS
NH2
N
N
ON
N
O
O
P
OO
P
O
HO
R
O
ATP
O
P
O
O
-
MOTOR CATALYZES
INTERACTION WITH
WATER TO CLEAVE
TERMINAL PHOSPHATE
BOND
OH
PROTEIN
O
H
+
O
O
PROTEIN
O
P O
O-
R
O
H
O
H
P
O
O
O
O
R O
H
ADP
+
O
H
P
O
O
PHOSPHATE
PROTEIN
PROTEIN
COORDINATION AT THE OPEN NUCLEOTIDE SITE
COORDINATION AT THE CLOSED NUCEOTIDE SITE
MYOSIN
KINESIN
NECK
3-HELIX
CYS-188
CONVERTER
DOMAIN
TRP-510
LOOP 9
SULFHYDRAL
HELIX
NUCLEOTIDE
SWITCH 2
HELIX
SWITCH 1
a3a
NUCLEOTIDE
SWITCH 2
P-LOOP
SWITCH 1
SWITCH 2
P-LOOP
ACTIN BINDING
SITE
MOVEMENT OF SWITCH 2
N-TERMINUS
G583
S551
O
G583
O
B
A
CONCLUSIONS
1. Comparison with myosin suggests a new structural state of
kinesin-family motors involving a closing of the nucleotide site.
2.
Spectroscopic data show the nucleotide site closes when kinesin
binds to microtubules.
3.
MD shows the open and closed binding-site conformations of
kinesin-family motors explain the changes in mobility of
nucleotide analog EPR probes upon binding to microtubules.
4. MD shows the closed state is essential for nucleotide hydrolysis
and force production.
COLLABORATORS
ROGER COOKE, UCSF
Nariman Naber
Marija Matuska
Kathy Franks-Skiba
RALPH YOUNT, WSU
Xiaoru Chen
Jean Grammer
PETER KOLLMAN, UCSF
Todd Minehardt
RON VALE, UCSF
Sarah Rice
ROBERTO CAR, PRINCETON
DAVID ADCOCK, LONE STAR BIOTECH