Transcript B - KIT

Structural investigations of the heterotetrameric E5/PDGF-receptor
β complex by oriented circular dichroism and solid state NMR
Dirk
1
Windisch ,
Claudia
1
Muhle-Goll ,
Stephan
1
Grage ,
1
Bürck ,
Jochen
Sergii
0°
90°
45°
90°
OCD [mdeg]
45°
200
300
Wavelenght [nm]
100
15N
ppm 300
200
100
15N
ppm 300
200
100
15N
ppm
theoretical solid-state 1D 15N-NMR spectra: the
shape and position of the signal (red) depends
on helix orientation. 15N powder tensor (black)
for comparison.
Oriented CD basis spectra: the
208 nm „fingerprint“
absorption band depends on
helix orientation.
1) structural investigations of PDGFR-TMD
di-C20:1 (DEiPC)
50000
C16:0/C18:1 (POPC)
D
E
16
12
Normalized OCD/ a.u.
B
di-C20:1 (DEiPC)
14
-10000
10
DEiPC
oriented
not oriented
C16:0/C18:0 (POPC)
75000
di-C20:1 (DEiPC)
7
di-C14:0 (DMPC)
65000
di-C18:1 (DOPC)
55000
DPC
POPC
4
2
0
DMPC
-2
-4
-40000
-6
190
260
45000
35000
25000
15000
5000
B
CONTIN
DEiPC
POPC
DMPC
DPC
α-Helix
65,2
63,9
64,9
74,4
S
h
e
6,7
4,4
5,1
0,1
e
t
t
u
r
n
9,9
10,9
11
10,6
a
n
d
o
m
N
18,2
20,8
18,9
14,6
R
M
S
the TMD is α-helical
C
D
0,045
0,048
0,049
0,031
A: CD analysis of the TMD reconstituted in
micelles and liposomes. The CD spectra
indicate an α-helical folding. B: Secondary
structure contents calculated using CONTIN.
The TMD has α-helical content of ~65% in
liposomes and of ~75% in micelles.
Ig
Ig
Ig
Ig
C: 1H15N 2D-HSQC spectrum of the TMD in DPC micelles with amino acid assignment.
D: Superposition of the 10 best NMR structures of the PDGFR-TMD dimer in DPC. The
TMD forms a left-handed coiled-coil with a dimer crossing angle of ~20°. The side
chains of Phe498 and Trp524 are shown in blue for visual orientation. E: Details of the
interface after a rotation of 30° around the z-axis. The contributing residues are
depicted in red and green, and the disordered termini are removed for clarity.
the TMD forms a left-handed coiled coil dimer
200
210
220
230
240
250
Ig
Ig
Ig
DEiPC
POPC
DMPC
DLPC
thickness
31,5 Å
27,1 Å
25,4 Å
20,9 Å
tilt
10°
17°
31°
n.d.
3
2
Ig
-S-S-S-S-
-S-S-S-S-
TK
TK
TK
TK
TK
TK
TK
TK
TK
C
C
C
C
E5
C
step-by-step strategy:
1) PDGFR-TMD dimer
2) E5 dimer
3) heterotetrameric E5/PDGFR-TMD complex
powder
0,04
0,26
0,54
>0,70
S
0,98
1
0,94
0,98
190
200
210
A: Oriented CD analysis of PDGFR-TMD in
macroscopically aligned lipid bilayers of
different thickness. The intesity increase
(DLPC->DMPC->POPC->DEiPC) of the 208 nm
band indicates a more upright orientation of
the TMD in thick membranes.
220
230
240
250
260
15N
ppm
B: Complementary solid-state NMR
analysis in oriented bilayers. The TMD is
much better reconstituted in thick
membranes. C: Summary solid state NMR
analysis: The TMD is well reconstituted
and less tilted in thick membranes.
β
CONTIN
DNPC
DErPC
DEiPC
DOPC
POPC
DPC
Helix
80%
78%
85%
60%
69%
85%
β-Strand
5%
8%
9%
5%
10%
3%
di-C18:1 (DOPC)
less tilted
2
1
270
more tilted
180
190
200
210
220
230
240
250
260
Wavelength [nm]
-
t
u
r
n
5%
6%
3%
11%
7%
5%
r
a
n
d
o
m
10%
8%
2%
24%
15%
9%
A: Oriented CD spectra of the E5 dimer in
macroscopically aligned lipid bilayers of
different thickness. The intesity increase
A: SR-CD analysis of E5 in micelles and
(DOPC -> DEiPC -> DErPC -> DNPC) of the
liposomes. The CD spectra indicate an α-helical 208 nm fingerprint band indicates a more
folding. B: Secondary structure contents
upright orientation of the E5 dimer in thick
calculated using CONTIN. The E5 protein has a membranes (DNPC and DErPC) and a more
higher α-helical content in thick lipid bilayers
tilted alignment in thin membranes (DOPC
and micelles (80-85%).
and DEiPC).
the TMD adjusts its tilt in response to lipid
the E5 protein is α-helical
bilayer thickness (to compensate hydrophobic
mismatch)
References
B
di-C20:1 (DEiPC)
3
-2
Wavelength [nm]
B
rmsd
0,0002
0,00024
0,0003
0,00031
di-C24:1 (DNPC)
4
-35000
180
260
next steps: investigations of the heterotetrameric E5/PDGFR-TMD complex
KIT – University of the State of Baden-Wuerttemberg and
National Research Center of the Helmholtz Association
1
TK
5
-1
Wavelength [nm]
r
Ig
Ig
6
-25000
DLPC
-
PDGF
0
-15000
more tilted
Wavelength [nm]
β
Ig
Ig
di-C22:1 (DErPC)
-5000
-30000
250
Ig
Ig
9
8
less tilted
6
di-C24:1 (DNPC)
di-C22:1 (DErPC)
di-C12:0 (DLPC)
8
95000
85000
-20000
240
Ig
-S-S-
A
0
230
Ig
A
10000
220
Ig
Ig
-S-S-
A
20000
210
Ig
-S-S-
2) orientation of E5 in lipid bilayers
DPC
30000
Ig
-S-S-
1) Secondary structure E5
di-C14:0 (DMPC)
40000
Ig
2) orientation of the TMD in lipid bilayers
2
MRE [deg*cm /dmol]
C
60000
Ig
2) structural investigations of E5
MRE [deg*cm²/dmol]
1) Secondary structure of the TMD in micelles and liposomes
70000
Ig
Ig
Dipolar Coupling
orientation in membranes
• solid-state NMR
N
N
1H15N
• Oriented CD (OCD)
The Platelet-derived growth factor receptor β is a member of the receptor-tyrosine-kinase family involved in development. The receptor can be activated
by two different ways: 1) The receptor is activated when the natural ligand PDGF binds simultaneously to the ectodomains of two receptors, resulting in
spatial proximity and subsequent cross-activation of the cytosolic kinase domains.1 2) The receptor can also be activated independently of the natural
ligand by binding of the small E5 oncoprotein from bovine papillomavirus.2 E5 is a small membrane protein of only 44 amino acids and it is thought to
manipulate the function of the receptor by specific helix-helix-contacts to the transmembrane of the receptor which then result in sustained receptor
activation and cell transformation.
To elucidate the helix-helix-interactions in receptor complex, we investigate the structure and membrane alignment of both proteins, first for each protein
and later in the heterotetrameric four-helix-bundle. For the structural analysis we combined circular dichroism (CD) spectroscopy and liquid-state NMR to
determine the secondary structure. Oriented CD and solid-state NMR were used to resolve the orientation of both proteins in their native environment.
Therefore the E5 protein and the transmembrane domain of the receptor (PDGFR-TMD: aa 494-531) were 15N-isotope labelled by bacterial protein
expression and reconstituted in detergent micelles and in oriented lipid bilayers. By CD and liquid-state NMR we found that both proteins are
predominantly α-helical in detergent micelles and in lipid bilayers.3,4 Furthermore, high resolution NMR measurements showed that PDGFR-TMD forms a
left-handed coiled-coil structure. A complementary OCD and solid-state NMR analysis of E5 and PDGFR-TMD reconstituted in different lipid bilayers
showed that the orientation of both proteins depends on the bilayer thickness, where both proteins were more tilted in thin membranes and less tilted in
thick membranes.
N
N
N
Normalized OCD/ a.u.
secondary structure
• high-resolution NMR
200
1,2
Ulrich
Ig
• (SR-) CD
190
Anne S.
Activation of the PDGF-receptor β
Tools
A
1
Afonin ,
Karlsruhe Institute of Technology, 1Institute for Biological Interfaces (IBG-2), POB 3640, 76021 Karlsruhe, Germany.
2Institute of Organic Chemistry and DFG Center for Functional Nanostructures, Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany.
contact: [email protected]
Poster #
0°
Silke
2
Hoffmann ,
15N
Chemical Shift
B: 2D solid-state NMR PISEMA analysis of the orientation
of E5 in magnetically aligned bicelles
(DMPC/DMPG[80:20]/6OPC; q=5). The weakly resolved
PISA-Wheel of E5 (black) can be superimposed with
theoretical PISA-Wheels of 0°-25° (with an order
parameter Smol = 0,9).
E5 adjusts its tilt in response to lipid bilayer thickness
(to compensate hydrophobic mismatch)