Mechanism of Signal Mechanism of Signal

Transduction by Transduction by

Rhodopsin as a Model Rhodopsin as a Model

GPCRGPCR

by by Basak IsinBasak Isin

OUTLINEOUTLINE

G Protein-Coupled-Receptors (GPCR)G Protein-Coupled-Receptors (GPCR) Aim of the projectAim of the project Gaussian Network Model (GNM)Gaussian Network Model (GNM) Anisotropic Network Model (ANM)Anisotropic Network Model (ANM) Results and DiscussionResults and Discussion Conclusion and SummaryConclusion and Summary Future PlansFuture Plans

GPCRsGPCRsthe largest superfamily of cell surface receptorsthe largest superfamily of cell surface receptors

seven helices - their signature motif 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.

RHODOPSINRHODOPSINthe first 3-dimensional molecular model for a GPCRthe first 3-dimensional molecular model for a GPCR

1

2 3

4

65

cytoplasmic region

extracellular region

7

8

located in the outer segments of rod photoreceptor cells in the retinaresponds to environmental signals, i.e., photons initiates intracellular processes that result in an electrical signal

processed by the visual system.

hCOOH

S-S-

VII

IVI

IIIV

V

NAc

VI V

VII

K

+

VI

III

COOH

S-S-

K

VII

IVI

IIIV

V

NAc

K

VI V

VII

III

Sensitization

Desensitization

LIGHT ACTIVATIONLIGHT ACTIVATION

11-cis retinal All-trans retinal

Metarhodopsin IIRhodopsin

Glycosylation at 2 asparagines 2 Cys(110/187) form sulfide bridgeLys296 at H7 is covalently attached to

11-cis retinal (protonated Schiff base) Glu113 at H3 is the counterion to Schiff

basePalmitate attached to 2 C-terminal CysSer334, 338 & 343 are major sites for

phosphorylation

SNAKE LIKE SNAKE LIKE REPRESENTATIONREPRESENTATION

the the Asp/Glu Arg Tyr (ERY)Asp/Glu Arg Tyr (ERY) motif at motif at the cytoplasmic end of the cytoplasmic end of helix 3helix 3

the the Asn Pro X X TyrAsn Pro X X Tyr (NPXXY)(NPXXY) motif motif in in helix 7helix 7 (Okada et al., 2002) (Okada et al., 2002)

the the X1BBX2X3BX1BBX2X3B motif at motif at cytoplasmic end of cytoplasmic end of helix 6helix 6 (B, basic; (B, basic; X, non-basic) (Ballesteros et al., X, non-basic) (Ballesteros et al., 1998),1998),

an ionic interaction between the an ionic interaction between the ligand and the receptor at the ligand and the receptor at the retinal retinal Schiff base Lys296Schiff base Lys296 and and the Schiff the Schiff base counterion the Glu113base counterion the Glu113 (Cohen et al., 1993),(Cohen et al., 1993),

the the Asn-AspAsn-Asp interaction between interaction between helices 1 and 2helices 1 and 2, respectively, respectively

the aromatic cluster surrounding the the aromatic cluster surrounding the ligand binding pockets (Visiers et al., ligand binding pockets (Visiers et al., 2002)2002)

MicrodomainsMicrodomains

AIM OF THE PROJECTAIM 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 ModelGaussian Network Model (GNM)(GNM)

For estimating the dynamic characteristics of For estimating the dynamic characteristics of

biomolecular structures based on atomic biomolecular structures based on atomic

coordinates in the native conformationcoordinates in the native conformation

Elastic network Virtual bond representationNo distinction between nonbonded and bonded neighborsThe interactions between residues in close proximity represented by harmonic potentials with a uniform spring constant

CONSTRUCTION OF CONSTRUCTION OF KIRCHHOFFKIRCHHOFF MATRIXMATRIX

5

1 2 3 4

18

19

2021

22

238

3

1 2 3 4……18 19 20 21….N

-1 -1 -1 -1…..-1 -1 -1…-1….0

7A

= U UT -1 = U -1UT

The fluctuations associated with kth mode

U: orthogonal (NxN) matrix

uk: kth eigenvector of Γ (1 ≤ k ≤ N) (shapes of corresponding mode of motion)

: diagonal matrix with eigenvalues (k)

1 = 0 <2 < …< N frequency of modes

= (3kBT/ ) (k-1[uk]i [uk]j )

the mechanism of the motion relevant to biological function.the mechanism of the motion relevant to biological function.

The maxima of the slow mode curves indicate the most flexible regions of the The maxima of the slow mode curves indicate the most flexible regions of the

molecule. molecule.

Identifies the hinge region that are important for biological function.Identifies the hinge region that are important for biological function.

Slow Modes Global Motions

a

222-230

6-11

27-33

26-37

14-20

cb d

Comparison of theoretically calculated all Comparison of theoretically calculated all modes with B- factors found by X-ray modes with B- factors found by X-ray

crystallographycrystallography

RESULTS AND DISCUSSION

Science, Palcwezski et. al, 2000

First Mode of GNMFirst Mode of GNM

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.01

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

Residue

Dis

tribu

tion

of F

luct

uaio

ns

H1

H2H3 H4

H5 H6 H7

12

76

53

The color codes are green, cyan, blue, magenta, pink and yellow in the order of

increasing mobility.

CROSS CORRELATIONCROSS CORRELATIONOf GNMOf GNM

Res

idue

num

ber

Residue number

a

Residue number

Residue number

Fast ModesFast ModesCritically important ones for the

overall stability of the molecule and

evolutionarily conserved

High Frequency ModesHigh Frequency Modes

Asn55

ANMANMthe three components of the inter-residue separation vectors obey the three components of the inter-residue separation vectors obey

Gaussian dynamics.Gaussian dynamics.involves the inversion of a 3N x 3N Hessian matrix involves the inversion of a 3N x 3N Hessian matrix HH that replaces the N that replaces the N

x N Kirchhoff matrix x N Kirchhoff matrix (Doruker (Doruker et al., et al., 20002000,, Atilgan Atilgan et al.,et al., 2001). 2001).

ANM results

Front View Back View

ANM results

Wild type

ab

17

6 3 5

28

8

1

8

5

6

241

72

63

5

12

34

6

5

7

7

4

MOVIESMOVIES

FRONTFRONT

BACKBACK

TWO CONFORMATIONSTWO CONFORMATIONS

1 2 34

55

678

4

321

678

b 1

11

2 22

33

3

4

4 4

55 5

6 6 67 7 7

8 8 8

wild type ANMbANMa

ANMa MM(199): E=-5531.2ANMb MM(273): E=-5235.41

Cross-sections from Cross-sections from the Topthe Top

Top-248

248-255

255-263

264-273

bottom

273-277

RRijij

H1

H2

H3

H4

H5

H6

H7H8

RETINAL REGIONRETINAL REGION

ALL ATOM MODELALL ATOM MODEL

•Adding the fluctutations of C to every atom in the PDB structure•Energy minimize the structure

•to see the side chain motions•to study the microdomains

E-MINE-MIN

NO RETINAL CIS-RETINAL TRANS-RETINAL

MICRODOMAIN1-ERYMICRODOMAIN1-ERY

ANM ANM WILD TYPEWILD TYPE

Wild Type 11-Trans Wild Type 11-Trans RetinalRetinal

CONCLUSIONCONCLUSION The rhodopsin ground state structure is predisposed for the functional The rhodopsin ground state structure is predisposed for the functional

conformational changes leading to the opening of the helical bundle, conformational changes leading to the opening of the helical bundle, thereby revealing the mechanism for this process.thereby revealing the mechanism for this process.

The mechanism for the observed opening of the helical bundle is The mechanism for the observed opening of the helical bundle is mediated by mediated by a torsional rotation of the molecule centered on Helix 3a torsional rotation of the molecule centered on Helix 3. .

The cytoplasmic end of helix 4 moves away from the cytoplasmic end of 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 helix 3, the most flexible region in helix 3, and stretches the cytoplasmic loop 2. cytoplasmic loop 2.

Helix 6 is rotates while simultaneously elongating, comparable to a Helix 6 is rotates while simultaneously elongating, comparable to a turning screw. turning screw.

Furthermore, they suggest a mechanism for the activation of the G-Furthermore, they suggest a mechanism for the activation of the G-protein. protein.

The screwing motion of helix 6 may provide a mechanical trigger for The screwing motion of helix 6 may provide a mechanical trigger for conformational changes in the G-protein which lead to GDP/GTP conformational changes in the G-protein which lead to GDP/GTP exchange.exchange.

SPECIFIC AIM SPECIFIC AIM 11A.A. The mechanism of activation of Rhodopsin by improved The mechanism of activation of Rhodopsin by improved GNM/ANM AnalysisGNM/ANM Analysis

1. Side Chain Interactions 1. Side Chain Interactions

2. Water Molecules 2. Water Molecules

3. Chromophore3. Chromophore

4. Missing loops 4. Missing loops

5. Lipid molecules 5. Lipid molecules

B. Chromophore binding pocketB. Chromophore binding pocket

C. Surface exposure after opening of the helical bundle C. Surface exposure after opening of the helical bundle during activationduring activation

D. Rhodopsin oligomerizationD. Rhodopsin oligomerization

(Fotiadis et al, 2003 Nature)(Fotiadis et al, 2003 Nature)

E. Analysis of multiple modes of the GNM and ANME. Analysis of multiple modes of the GNM and ANM

SPECIFIC AIM SPECIFIC AIM 22Interaction of rhodopsin with GInteraction of rhodopsin with Gtt

Analysis of GDP Analysis of GDP Bound form of Bound form of Heterotrimeric GHeterotrimeric Gt t by by GNM and ANM.GNM and ANM.

interaction surface for interaction surface for rhodopsin- Grhodopsin- Gtt complex by complex by electrostatic surface maps electrostatic surface maps of the active form of of the active form of rhodopsin found by ANM rhodopsin found by ANM analysis and Ganalysis and Gtt. .

SPECIFIC AIM SPECIFIC AIM 33

by exploring the importance of hinge regions by by exploring the importance of hinge regions by sequence alignment. sequence alignment.

by applying the GNM and ANM analysis to other by applying the GNM and ANM analysis to other members of the family whose structures are members of the family whose structures are determined theoretically.determined theoretically.

Extension of the mechanism of Activation to other GPCRsExtension of the mechanism of Activation to other GPCRs

Adrenergic receptorsAdrenergic receptorsMetabotropic Glutamate receptorsMetabotropic Glutamate receptors

Thanks toThanks toDr. Ivet BaharDr. Ivet Bahar Dr. Judith KleinDr. Judith Klein

-Seetharaman-Seetharaman

Post-Docs:Dr. Rajan MunshiDr. Dror Tobi

Dr. A.J. Rader

Graduate StudentsElife Zerrin BagciShann-Ching ChenChris Myers Alpay TemizLee-Wei Yang

Dr Mike Cascio Dr Billy DayDr Christine MilcarekDr Hagai MeirovitchDr Tom Smithgall

System AdministratorsDr. Rob Bell Mark Holliman AdministratorsJoseph BaharNancy Gehenio

Summary and ConclusionSummary and ConclusionThe 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 calculationsExperiments to validate the calculations

Site directed spin labeling combined with EPRSite 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 calculationsExperiments to validate the calculationsRhodopsin-Gt InteractionRhodopsin-Gt Interaction

Gt activation by fluoresence spectroscopy: After GTPS 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.