Transcript isin-mar-2004

Mechanism of Signal
Transduction by Rhodopsin as a
Model GPCR
by
Basak Isin
OUTLINE
G Protein-Coupled-Receptors (GPCR)
 Aim of the project
 Gaussian Network Model (GNM)
 Anisotropic Network Model (ANM)
 Results and Discussion
 Conclusion and Summary
 Future Plans

GPCRs
the largest superfamily of cell surface receptors
seven helices - their signature motif
involved in a number of clinically important
ligand/receptor processes.
bind ligands from the cell exterior, which induce a
conformational change in the cytoplasmic face of the
receptor, enabling binding of the G protein.
couple to heterotrimeric G proteins to convert an
extracellular signal into an intracellular signal.
significant drug targets. 50-60% of approved drugs target
members of the GPCR family.
RHODOPSIN
the first 3-dimensional molecular model for a GPCR
cytoplasmic
region
8
4
1
2
6
3
5
7
extracellular
region
located in the outer segments of rod photoreceptor cells in the retina
responds to environmental signals, i.e., photons
initiates intracellular processes that result in an electrical signal processed by the visual
system.
LIGHT ACTIVATION
Rhodopsin
11-cis retinal
Metarhodopsin II
All-trans retinal
SNAKE LIKE REPRESENTATION
Glycosylation at 2 asparagines
2 Cys(110/187) form sulfide bridge
Lys296 at H7 is covalently attached to
11-cis retinal (protonated Schiff base)
Glu113 at H3 is the counterion to Schiff
base
Palmitate attached to 2 C-terminal Cys
Ser334, 338 & 343 are major sites for
phosphorylation
Microdomains






the Asp/Glu Arg Tyr (ERY) motif at the
cytoplasmic end of helix 3
the Asn Pro X X Tyr (NPXXY) motif in
helix 7 (Okada et al., 2002)
the X1BBX2X3B motif at cytoplasmic end
of helix 6 (B, basic; X, non-basic) (Ballesteros
et al., 1998),
an ionic interaction between the ligand and
the receptor at the retinal Schiff base
Lys296 and the Schiff base counterion the
Glu113 (Cohen et al., 1993),
the Asn-Asp interaction between helices 1
and 2, respectively
the aromatic cluster surrounding the ligand
binding pockets (Visiers et al., 2002)
AIM OF THE PROJECT
Understanding the mechanism of activation of
rhodopsin as a model GPCR and its interaction
with Gt by structure-functions analysis using the
GNM and its extension, the ANM.
Gaussian Network Model (GNM)
For estimating the dynamic characteristics of biomolecular
structures based on atomic coordinates in the native conformation
Elastic network
Virtual bond representation
No distinction between nonbonded and bonded neighbors
The interactions between residues in close proximity
represented by harmonic potentials with a uniform spring
constant
CONSTRUCTION OF KIRCHHOFF MATRIX
19
20
18
21
22
1
2
3
4……18
19
20 21….N
1
2
8
3
7A
4
23
5
3
-1
-1
-1
-1…..-1
-1
-1…-1….0
G = U L UT

G-1 = U L-1UT
The fluctuations associated with kth mode
= (3kBT/ ) (lk-1[uk]i [uk]j )
U: orthogonal (NxN) matrix
uk: kth eigenvector of Γ (1 ≤ k ≤ N) (shapes of corresponding mode of motion)
L: diagonal matrix with eigenvalues (lk)
l1 = 0 < l2 < …< lN frequency of modes
Global Motions
Slow Modes
 the mechanism of the motion relevant to biological function.
 The maxima of the slow mode curves indicate the most flexible regions of the
molecule.
 Identifies the hinge region that are important for biological function.
a
2733
622211
230
b
c
2637
1420
d
RESULTS AND DISCUSSION
Comparison of theoretically calculated all modes with Bfactors found by X-ray crystallography
Science, Palcwezski et. al, 2000
First Mode of GNM
0.01
0.009
Distribution of Fluctuaions
0.008
0.007
1
2
7
0.006
3
6
5
0.005
0.004
0.003
0.002
H2
0.001
H5
H3
H1
H6
H7
H4
0
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
Residue
The color codes are green, cyan, blue, magenta, pink and yellow in the order of
increasing mobility.
340
CROSS CORRELATION
Of GNM
a
Residue number
R
esi
du
e
nu
mb
er
Residue number
Residue number
Fast Modes
High Frequency Modes
Asn55
Critically important ones for the
overall stability of the molecule and
evolutionarily conserved
ANM
the
three components of the inter-residue separation vectors obey Gaussian dynamics.
involves
the inversion of a 3N x 3N Hessian matrix H that replaces the N x N Kirchhoff
matrix G (Doruker et al., 2000, Atilgan et al., 2001).
a
Front View
8
7
6
3
5
1
7
2
5
3
4
8
2
6
4
b
6
6
8
2
1
Back View
7
2 1
1
5
5
4
3
7
ANM results
ANM results
Wild type
MOVIES
FRONT
BACK
TWO CONFORMATIONS
8
8
8
4
3
3
1
7
2
6
4
7
5
1
ANMa
4
5
6
2
wild type
ANMa MM(199): E=-5531.2
ANMb MM(273): E=-5235.41
5
1
7
2
6
3
ANMb
b
1
8
2
7
4
4
3
2
1
3
5
6
5
8
7
6
Cross-sections from the Top
Top-248
248-255
255-263
264-273
273-277
bottom
Rij
H1
H2
H3
H4
H5
H6
H7
H8
RETINAL REGION
ALL ATOM MODEL
•to see the side chain motions
•to study the microdomains
•Adding the fluctutations of C to every atom
in the PDB structure
•Energy minimize the structure
E-MIN
NO RETINAL
CIS-RETINAL
TRANS-RETINAL
MICRODOMAIN1-ERY
ANM
WILD TYPE
Wild Type 11-Trans Retinal
CONCLUSION

The rhodopsin ground state structure is predisposed for the functional conformational
changes leading to the opening of the helical bundle, thereby revealing the mechanism
for this process.

The mechanism for the observed opening of the helical bundle is mediated by a
torsional rotation of the molecule centered on Helix 3.

The cytoplasmic end of helix 4 moves away from the cytoplasmic end of helix 3, the
most flexible region in helix 3, and stretches the cytoplasmic loop 2.

Helix 6 is rotates while simultaneously elongating, comparable to a turning screw.

Furthermore, they suggest a mechanism for the activation of the G-protein.

The screwing motion of helix 6 may provide a mechanical trigger for conformational
changes in the G-protein which lead to GDP/GTP exchange.
SPECIFIC AIM 1
A. The mechanism of activation of Rhodopsin by improved GNM/ANM
Analysis
1. Side Chain Interactions
2. Water Molecules
3. Chromophore
4. Missing loops
5. Lipid molecules
B. Chromophore binding pocket
C. Surface exposure after opening of the helical bundle during activation
D. Rhodopsin oligomerization
(Fotiadis et al, 2003 Nature)
E. Analysis of multiple modes of the GNM and ANM
SPECIFIC AIM 2
Interaction of rhodopsin with Gt

Analysis of GDP Bound
form of Heterotrimeric Gt by
GNM and ANM.
interaction
surface for rhodopsinGt complex by electrostatic surface
maps of the active form of
rhodopsin found by ANM analysis
and Gt.
SPECIFIC AIM 3
Extension of the mechanism of Activation to other GPCRs


by exploring the importance of hinge regions by sequence
alignment.
by applying the GNM and ANM analysis to other members of
the family whose structures are determined theoretically.
Metabotropic Glutamate receptors
Adrenergic receptors
Thanks to
Dr. Ivet Bahar
Dr. Judith Klein
-Seetharaman
Post-Docs:
Dr. Rajan Munshi
Dr. Dror Tobi
System Administrators
Dr. Rob Bell
Mark Holliman
Administrators
Joseph Bahar
Nancy Gehenio
Dr. A.J. Rader
Graduate Students
Elife Zerrin Bagci
Shann-Ching Chen
Chris Myers
Alpay Temiz
Lee-Wei Yang
Dr Mike Cascio
Dr Billy Day
Dr Christine Milcarek
Dr Hagai Meirovitch
Dr Tom Smithgall
Summary and Conclusion
The elastic network models (GNM and ANM) are tools to explore the dynamics of proteins
and to determine the critically important sites. These are classified in two categories:



The first category comprises the residues that are important for
coordinating the cooperative motions of the overall molecule.
These are identified from the minima of the global mode shapes.
Their mutation can impede function.
The second one consists of residues experiencing an extremely
strong coupling to their close neighbors, and thereby undergoing
the highest frequency/smallest amplitude vibrations.
Their mutation can impede stability.
Both groups of residues are expected to be evolutionarily
conserved, the former for function requirements, and the latter for
folding and stability
Experiments to validate the calculations

Site directed spin labeling combined with EPR
Cysteine scaning mutagenesis:
-Reactivity and solvent accesibility of the sulfhydryl groups to 4-PDS. Absorbance of 4-TP at
323nm
-Disulfide exchange of thiopyridinyl-derivatives of rhodopsin by sulfhydryl reagents (R-SH) both
in dark and after illumination.
-Disulfide bond formation in double cysteine mutation assay. Rate of cysteine bond formation is a
measure of proximity of the mutant. In addition, sulfur bridges can inhibit light activation. This
can show the necessary movements of helices to form MetaII.
Site directed 19F labeling for NMR study: single cysteine mutants followed by the attachment of
TET (CF3-CH2-S) attachment. Shifts in dark and light NMR spectra of the mutants shows the
movements of residues.
Antibody binding experiments: Antibodies which bind to Meta II but not rhodopsin in the dark.
Experiments to validate the calculations
Rhodopsin-Gt Interaction




Gt activation by fluoresence spectroscopy: After GTPS addition, increase in
fluoresence results from exposure of Trp207 in Gt. when Gt is activated.
Assay of Meta II-Gt complex: Flash photolysis (light scattering). A flash
induced light scattering increases over time in the presence of binding but not
in the absence of Gt. This signal reflects the binding of Gt to R. In the
presence of Gt and GTP, a flash produces a decrease of scattering intensity
due to a loss of scattering mass.
Nucleotide release assay for GDP release ability. Samples are filtered through
a nitrocellulose membrane. The amount of [32P]GDP is filtered and
quantitated.
Peptide competition assays.