G PROTEIN COUPLED RECEPTORS - PowerPoint Presentation

Slide1

G PROTEIN COUPLED RECEPTORS

Slide2

GPCR FAMILY

CLASS A STRUCTURAL ANALYSIS

TASTE RECEPTORS

CONCLUSIONS & QUESTIONS

Slide3

GPCRS. OVERVIEW

Also known as

7TM receptors

Largest family of proteins in the human genome (Nearly 1000 such receptors are though to be present )

Mediate signal transduction by recognizing different stimuli such as photons of light, biogenic amines, peptides….

Mediates responses to visual, olfactory, hormonal, neurotransmitter and others…

Involved in many different diseases so half of the drug targets in the pharmaceutical industry are GPCRs

Slide4

Membrane proteins with seven

transmembrane

domains

Upon activation, signal gets transmitted to the

cytoplasmatic

face and amplifies through

heterotrimeric

G protein complex

Slide5

Slide6

GPCRS.

OVERVIEW (II)

Very hard-to-

crystalize

proteins

First high resolution

cristal

was

Rhodopsin

Currently just four groups of proteins have an available PDB structure

Three differentiated regions: extracellular,

transmembrane

and

intracelullar

Slide7

Slide8

GPCRS. STRUCTURAL OVERVIEW (III)

There is a large gap in experimental GPCR structural space

Currently just 5 groups of GPCRs structurally solved

ADENOSINE-2A RECEPTOR

β-1 ADRENERGIC RECEPTOR

β-2 ADRENERGIC RECEPTOR

RHODOPSIN

RHODOPSIN

(ALL OF THEM BELONGING TO CLASS A GPCRs)

Slide9

GPCRs

CLASS A - STRUCTURAL ANALYSIS

Slide10

CLASS A FAMILY OVERVIEW

SEQUENCE SIMILARITIES. CONSERVED MOTIFS

STRUCTURAL ANALYSIS

EXTRACELLULAR REGION

LIGAND BINDING POCKET (TRANSMEMBRANE)

INTRACELLULAR REGION

CONCLUSIONS & QUESTIONS

Slide11

Main common regions:

N-terminus

Extracellular loops (ECL1, 2, 3

)

Transmembrane

Helices (

TMH1, 2, 3, 4, 5, 6, 7,8

)

Intracellular loops (

ICL1, 2, 3

)

C-terminus

Some structural features are shared by all

Pro distortions in TMHs 4,5,6 and 7

Disulphide bridge between TMH3 and ECL2Some other features are either unique to a particular receptor or shared by a subset (i.e specific loop conformation)The most distinct features are observed in the extracellular and intracellular loops

CLASS A - STRUCTURAL ANALYSIS

Slide12

GPCRS. STRUCTURAL OVERVIEW

GRAFS

system

considers

five

main

families

:

GLUTAMATE

(

G

) (CLASS C*)RHODOPSIN (R) (CLASS A*)ADHESION (A) (CLASS B*)FRIZZLED/TASTE2 (F) (FRIZZLED CLASS*)SECRETIN (S) (CLASS B*)* NC-IUPHAR NOMENCLATURE SYSTEM

Slide13

CLASS A - STRUCTURAL ANALYSIS

PDBs used as representative structures in the structural analysis:

ADENOSINE-2A RECEPTOR (

Human

):

3EML

β-1 ADRENERGIC RECEPTOR (

Turkey

):

2VT4

β-2 ADRENERGIC RECEPTOR (

Human

):

2RH1

RHODOPSIN (Squid): 2Z73 RHODOPSIN (Bovine): 1U19

Slide14

Comparison of amino acid sequences of these receptors reveal modest conservation ranging from

22%

to 64% sequence identity

CLASS A - STRUCTURAL ANALYSIS

Slide15

SQUID

RHODOPSIN

BOVINE

RHODOPSIN

ADENOSINE

2A RECEPTOR

β-1 ADREN. RECEPTOR

β-2 ADREN.

RECEPTOR

SQUID RHODOPSIN

27%

22%

25%

25%

BOVINE

RHODOPSIN

27%

22%

24%

23%

ADENOSINE2A RECEPTOR

22%22%36%33%β-1 ADREN. RECEPTOR 25%24%36%64%β-2 ADREN. RECEPTOR25%23%33%64%

CLASS A - STRUCTURAL ANALYSIS

Percentage of sequence identity within receptors

Slide16

Comparison of amino acid sequences of these receptors reveal modest conservation ranging from

22%

to 64% sequence identity

When restricting the comparison to individual helices, differences in sequence similarity between each receptor are higher (although still small…)

CLASS A - STRUCTURAL ANALYSIS

MSA of the firs

Transmembrane

Helix I (TMH1) of all 5 receptors

Slide17

CLASS A - STRUCTURAL ANALYSIS

MSA of the five receptors structurally solved identified 25 conserved residues:

Slide18

Conserved segments are localized in the

transmembrane

domains, among them the most highly conserved are:

E/DRY

motif in TMH3

CLASS A - STRUCTURAL ANALYSIS

MSA of

Transmembrane

Helix III (TMH3) of all 5 receptors

Slide19

WXPF/Y

motif in TMH6

CLASS A - STRUCTURAL ANALYSIS

MSA of

Transmembrane

Helix VI (TMH6) of all 5 receptors

Slide20

NPXIY

motif in TMH7

CLASS A - STRUCTURAL ANALYSIS

MSA of Helix VII (TMH7) of all 5 receptors

Slide21

CLASS A - STRUCTURAL ANALYSIS

β-2 ADRENERGIC RECEPTOR

RHODOPSIN (

Bovine

)

ADENOSINE-2A RECEPTOR)

RHODOPSIN (

Squid

)

β-1 ADRENERGIC RECEPTOR

Slide22

CLASS A - STRUCTURAL ANALYSIS

Structural

superpositioning

of the 5 receptors demonstrating a high level of overall structure similarity

Slightly more variation at the extracellular side of the membrane surface

RMSDs of superimposition ranging from 0.63Å to 4.03Å

Slide23

CLASS A - STRUCTURAL ANALYSIS

EXTRACELLULAR REGION

RHODOPSIN

Extensive secondary and tertiary structure to completely occlude the binding site from solvent access (“retinal plug”)

N-terminus along with ECL2 form a four-stranded

β

-

sheet with additional interactions

ECL3-ECL1

Access to retinal binding pocket severely restricted

Slide24

Slide25

CLASS A - STRUCTURAL ANALYSIS

N-TERMINUS

ECL-2

ECL-1

ECL-3

Slide26

CLASS A - STRUCTURAL ANALYSIS

EXTRACELLULAR REGION

RHODOPSIN

Extensive secondary and tertiary structure to completely occlude the binding site from solvent access (“retinal plug”)

N-terminus along with ECL2 form a four-stranded

β

-

sheet with additional interactions

ECL3-ECL1

Access to retinal binding pocket severely restricted

One disulfide bridge (it has been shown to be essential for the normal function of

Rhodopsin

)

Slide27

CLASS A - STRUCTURAL ANALYSIS

CYS 187 (ECL2)

CYS 110 (TMH3)

Slide28

CLASS A - STRUCTURAL ANALYSIS

Slide29

CLASS A - STRUCTURAL ANALYSIS

Β

-ADRENERGIC RECEPTORS

Extracellular region much more open

Short helical segment within ECL2:

Limited interactions with ECL1

2 disulfide bridges: one with a coil segment of ECL2 and the other fixing the entire loop to the top of TMH3

The random coil section of ECL2 forms the top of the ligand binding pocket (only partially occluded)

ECL3 forms no interaction with ECL1 or ECL2

Slide30

CLASS A - STRUCTURAL ANALYSIS

CYS 190 (ECL2)

CYS 184 (ECL2)

CYS 106 (TMH3)

CYS 191 (ECL2)

Slide31

CLASS A - STRUCTURAL ANALYSIS

Β

-ADRENERGIC

Extracellular region much more open

Short helical segment within ECL2:

Limited interactions with ECL1

2 disulfide bridges: one with a coil segment of ECL2 and the other fixing the entire loop to the top of TMH3

The random coil section of ECL2 forms the top of the ligand binding pocket (only partially occluded)

ECL3 forms no interaction with ECL1 or ECL2

Entire 28-resiude N -terminus completely disordered in the four structures solved to date

Does the extracellular region of the β-Adrenergic family has evolved to allow access to the ligand binding site?

Slide32

CLASS A - STRUCTURAL ANALYSIS

RHODOPSIN

Β

-ADRENERGIC RECEPTOR

?

Slide33

CLASS A - STRUCTURAL ANALYSIS

ADENOSIN RECEPTORS

Highly constrained by four disulfide bridges and multiple ligand binding interactions

Three out of the four disulfide bridges constrain the position of ECL2 anchoring this loop to ECL1 and the top of TMH3

Slide34

CLASS A - STRUCTURAL ANALYSIS

CYS 262 (TMH6)

CYS 259 (ECL3)

CYS 71(ECL1)

CYS 159 (ECL2)

CYS 166 (ECL2)

CYS 77 (TMH3)

CYS 74

(TMH3)

CYS 146 (N-TERMINUS)

Slide35

CLASS A - STRUCTURAL ANALYSIS

ADENOSIN RECEPTORS

Highly constrained by four disulfide bridges and multiple ligand binding interactions

Three out of the four disulfide bridges constrain the position of ECL2 anchoring this loop to ECL1 and the top of TMH3

The former three disulfide bridges probably stabilize a short helical segment N terminal of TMH5 containing

Phe168 and Glu169

. This segment is considered to be an important region for ligand binding

Slide36

CLASS A - STRUCTURAL ANALYSIS

DISULFIDE BRIDGES

PHE 168

GLU 169

RANDOM COIL (ECL2)

Slide37

CLASS A - STRUCTURAL ANALYSIS

DISULFIDE BRIDGE

PHE 168

GLU 169

RANDOM COIL (ECL2)

Slide38

CLASS A - STRUCTURAL ANALYSIS

ADENOSIN RECEPTORS

Highly constrained by four disulfide bridges and multiple ligand binding interactions

Three out of the four disulfide bridges constrain the position of ECL2 anchoring this loop to ECL1 and the top of TMH3

The former three disulfide bridges probably stabilize a short helical segment N terminal of TMH5 containing

Phe168 and Glu169

. This segment is considered to be an important region for ligand binding

ECL3 contains another disulfide bridge that might constrain His264 position, which in turn forms a polar interaction with Glu169

Slide39

CLASS A - STRUCTURAL ANALYSIS

LIGAND BINDING POCKET

RHODOPSIN (I)

11-cis-retinal is covalently bound to Lys296 in TMH7 by a

protonated

Shiff

base

This ligand stabilizes the inactive state of

rhodopsin

until photon absorption occurs.

Slide40

Slide41

CLASS A - STRUCTURAL ANALYSIS

LIGAND BINDING POCKET

RHODOPSIN (I)

11-cis-retinal covalently bound to Lys296 in TMH7 by a

protonated

Shiff

base. This ligand stabilizes the inactive state of

rhodopsin

until photon absorption

The

molecular

switch

involved in the activation of the receptor

is

a

is

a

rotamer

toogle

switch The indole chain of the highly conserved W265 is in van der Waals contact with the β-ionone ring of retinal

Slide42

11-CIS-RETINAL

W265 (

Toggle

switch

)

Slide43

CLASS A - STRUCTURAL ANALYSIS

11-CIS-RETINAL

Slide44

CLASS A - STRUCTURAL ANALYSIS

Slide45

CLASS A - STRUCTURAL ANALYSIS

TRP265

LYS 296

PHE 261

PHE 212

MET207

TYR191

GLU 181

GLU 113

Slide46

CLASS A - STRUCTURAL ANALYSIS

LIGAND BINDING POCKET

RHODOPSIN (II)

Binding pocket comprises a cluster of the following residues: Glu113, Glu181, Tyr191, Met207, Phe212, Phe261, Phe293, Lys296 and Trp265

The position of this binding pocket does not vary too much between different subspecies

Prior to activation, a chained series of conformational changes occur. Among this changes, it’s worth highlighting that Lys296 releases from ligand

Slide47

CLASS A - STRUCTURAL ANALYSIS

LYS 296

11-CIS-RETINAL

TRP265

Slide48

CLASS A - STRUCTURAL ANALYSIS

LIGAND BINDING POCKET

RHODOPSIN (III)

Binding pocket comprises a cluster of the following residues: Glu113, Glu181, Tyr191, Met207, Phe212, Phe261, Phe293, Lys296 and Trp265

The position of this binding pocket does not vary too much between different subspecies

An extended hydrogen-bonded network (

ionic lock

) between TMH3 and TMH6 is present. Breakage of this ionic lock needs to happen for receptor’s activation

Slide49

CLASS A - STRUCTURAL ANALYSIS

BINDING POCKET

GLU134

THR251

GLU 247

IONIC LOCK

ARG135

TMH6

TMH3

Slide50

CLASS A - STRUCTURAL ANALYSIS

β

-ADRENERGIC RECEPTORS

Similar binding pocket to the

Rhodopsin’s

one, position does not vary considerably with alternate

ligands

or between different species (

Hanson et al.2008; Warne et al.2008

)

As a representative ligand,

carazolol

follows a similar path as that of

rhodopsin

Slide51

CLASS A - STRUCTURAL ANALYSIS

CARAZOLOL

W286 (

Toggle

switch

)

Slide52

CLASS A - STRUCTURAL ANALYSIS

Slide53

CLASS A - STRUCTURAL ANALYSIS

β

-ADRENERGIC RECEPTORS

Similar binding pocket to the

Rhodopsin’s

one, position does not vary considerably with alternate

ligands

or between different species (

Hanson et al.2008; Warne et al.2008

)

β-adrenergic

ligands

interact with the receptor through two cluster of polar interactions:

Slide54

CLASS A - STRUCTURAL ANALYSIS

SER203

ASN312

SER207

SER204

TYR316

Slide55

CLASS A - STRUCTURAL ANALYSIS

β

-ADRENERGIC RECEPTORS

Similar binding pocket to the

Rhodopsin’s

one, position does not vary considerably with alternate

ligands

or between different species (

Hanson et al.2008; Warne et al.2008

)

As a representative ligand,

carazolol

follows a similar path as that of

rhodopsinβ-adrenergic ligands interact with the receptor through two cluster of polar interactions:Positively charged secondary amine group and β-OH interact with Tyr316 in TMH3 and two asparagines on TMH7

Slide56

CLASS A - STRUCTURAL ANALYSIS

ASN312

CLUSTER OF SERINES

ASN113

TYR316

Slide57

CLASS A - STRUCTURAL ANALYSIS

β

-ADRENERGIC RECEPTORS

Similar binding pocket to the

Rhodopsin’s

one, position does not vary considerably with alternate

ligands

or between different species (

Hanson et al.2008; Warne et al.2008

)

As a representative ligand,

carazolol

follows a similar path as that of

rhodopsinβ-adrenergic ligands interact with the receptor through two cluster of polar interactions:Positively charged secondary amine group and β-OH interact with Tyr216 in TMH3 and two asparagines on TMH7The second group comprises a cluster of serine residues on TMH5

Slide58

CLASS A - STRUCTURAL ANALYSIS

SER203

SER207

SER204

TRP286

Slide59

CLASS A - STRUCTURAL ANALYSIS

ADENOSIN 2A

With the recent elucidation of this structure (2008), we see a very different location of the binding pocket

Slide60

CLASS A - STRUCTURAL ANALYSIS

ZM241385

W246(

Toggle

switch

)

Slide61

CLASS A - STRUCTURAL ANALYSIS

ADENOSINE 2A

With the recent elucidation of this structure (2008), we see a very different location of the binding pocket

This pocket changes in position and orientation with respect to both

rhodopsin

and adrenergic receptors

Slide62

CLASS A - STRUCTURAL ANALYSIS

Slide63

CLASS A - STRUCTURAL ANALYSIS

TRP246

Slide64

CLASS A - STRUCTURAL ANALYSIS

ADENOSINE 2A

With the recent elucidation of this structure (2008), we see a very different location of the binding pocket

This pocket changes in position and orientation with respect to both

rhodopsin

and adrenergic receptors

Adenosin

ligand ZM241385 forms mainly polar interactions with THM5

Slide65

CLASS A - STRUCTURAL ANALYSIS

TRP246

TMH5

Slide66

CLASS A - STRUCTURAL ANALYSIS

ADENOSINE 2A

With the recent elucidation of this structure (2008), we see a very different location of the binding pocket

This pocket changes in position and orientation with respect to both

rhodopsin

and adrenergic receptors

Adenosin

ligand ZM241385 forms mainly polar interactions with THM5

But

ECL2

also plays an important role in binding affinity, through interacting with Glu169 and Phe168

Slide67

CLASS A - STRUCTURAL ANALYSIS

PHE168

GLU169

ECL2

Slide68

CLASS A - STRUCTURAL ANALYSIS

INTRACELLULAR REGION

The so called “ionic lock” that we saw for

rhodopsin

was though to be conserved in the region formerly described as

DRY motif

The determination of adrenergic and adenosine receptors demonstrate no universality of the ionic lock among class A receptors

The

DRY

motif interacts with

ICL2

through a polar interaction between the ASP and SER/TYR on ICL2

DRY

interaction is still though to play a key role in linking the conformational changes that take place upon agonist binding to the downstream effects

Slide69

CLASS A - STRUCTURAL ANALYSIS

TYR112

ASN102

ASN101

DRY

TYR103

ICL2

ADENOSINE RECEPTOR

Slide70

CLASS A - STRUCTURAL ANALYSIS

CONCLUSIONS

Extracellular and intracellular regions show more diversity

Conserved disulfide bridges

stabilise

extracellular domain

Transmembrane

region is more structurally conserved

TRP acts as

toogle

switch

rotamer

and is conserved in all structures solved to date

Ionic lock theory just valid for

RhodopsinDRY motif conserved throughout but functions remain still not fully knwon

Slide71

CASE STUDY:

TASTE RECEPTORS

Slide72

TASTE RECEPTORS OVERVIEW

CONSERVATION

MODELING

STRUCTURE

CONCLUSIONS

Slide73

Five basic tastes:

Salty

SourBitter

Umami

Sweet

Sweet and

Umami

related with appetitive sensations

Bitter sense related to the rejection of food

TASTE RECEPTORS

Ligand

-gated

cation

channels

G protein-coupled receptors

The most important for food acceptance

Slide74

Sweet receptors evolved to accept sugars, because the glucose is the source of energy of the organism.

Umami

receptors to recognize proteins sources like peptides or aminoacids.

The bitter ones to avoid ingestion of toxic compounds, mainly from plants.

TASTE RECEPTORS

Slide75

Sweet and

umami

senses are mediated by three C class GPCRs: T1R1, T1R2 & T1R3. These receptors have the characteristic 7 helix TM domain and a large extracellular domain with the Venus Flytrap (VFT) that contains the active site for typical

ligands

.

The receptors combine as

heterodimers

:

The T1R2-T1R3 is the sweet receptor whereas the T1R1-T1R3 acts as the

aminoacid

receptor which gives the

umami

taste.

The sweet receptor can recognize a wide range of molecules (carbohydrates,

aminoacids, peptides…) because have several active sites.SWEET AND UMAMI

Slide76

Agonists:

Sucrose, fructose,

galactose, glucose, lactose, maltose. Amino acids like glycine

, D-tryptophan, glutamate, the sweet proteins

brazzein

,

monellin

and

traumatin

. And the synthetic sweeteners cyclamate, saccharin,

acesulfame

K, aspartame,

dulcin

,

neotame and sucraloseAntagonists:Lactisole.SWEET RECEPTOR (T1RS/T1R3) LIGANDS

Slide77

T1RS RECEPTORS

Slide78

Bitter

A large family (~30 members) of class A GPCR.

Known as T2Rs.

Each receptor can

recognise

a wide variety of bitter molecules.

These group of receptors lack the large N-terminal extracellular domain but may act as

dimers

as well.

Slide79

Bitter

Slide80

Since we cannot compare the structures of the

differents

proteins of this group we will study the sequence conservation within each protein and between the different proteins.We have performed multiple alignments using T-COFFE and

Jalview

to get some additional features.

T1RS CONSERVATION

Slide81

T1R1:

Only Mouse, Rat and Human have this protein.

By evolutionary terms not understandable why these three species.

Probably lack of annotation in primates and other species would be a reason.

Almost perfectly conserved. (99 out of 100)

T1RS CONSERVATION

Slide82

T1R3:

Human, Rat, Mouse, Primates(Chimpanzee and Gorilla) and Dog and Cat.

Again the lack of annotation of this protein may result in these few species.

Almost perfectly conserved. (99 out of 100)

T1RS CONSERVATION

Slide83

T1R2:

The most characteristic sweet taste receptor

Eight species of primates, rat, mouse, cat and dog have this protein annotated.

Worst score for this protein but still highly conserved. (93 out of 100)

It may be an artifact due to have more sequences.

T1RS CONSERVATION

Slide84

T1Rs Signal

The peptide signal to export the protein to the membrane.

Low conservation.Each member of the family may have a different signal because should be in specific positions in the membrane.

T1RS CONSERVATION

Slide85

T1Rs Venus Flytrap (VFT)

Good general conservation.

Loop regions with more variability.

T1RS CONSERVATION

Slide86

T1Rs Venus Flytrap (VFT)

T1RS CONSERVATION

Slide87

T1Rs Venus Flytrap (VFT)

T1RS CONSERVATION

Slide88

T1Rs

Cysteine

Rich Domain:As expected the Cysteins are conserved in all the members of the family.

Polar (Serine, Glutamine, Tryptophan,

Histidine

) and Aspartic acid well conserved, this region have as well some binding affinity to

ligands

.

T1RS CONSERVATION

Slide89

T1Rs

Tansmembrane

Domain:

T1RS CONSERVATION

Slide90

T1Rs Phylogeny:

From the global alignment of the entire dataset, a

phylogenetic tree were performed.

Obviously is clustered in the three families as expected, the three different proteins.

Primates and rodents clustered.

Again, family discovered in 2001, therefore there is lack of annotation in a lot of species.

T1RS CONSERVATION

Slide91

T1RS CONSERVATION

Slide92

No crystal structure solved yet.

Homology models built from the known extracellular structures of

Metabotropic Glutamate Receptors and crystal transmembrane

domains from class A GPCRs.

We have performed a homology model basing on these known structures.

T1RS MODELING

Slide93

T1RS MODELING

Slide94

Crucial points:

Manual refinement

Most of the cysteins in the alignment were misaligned.

Built two different models for each protein of the

heterodimer

(T1R2 & T1R3)

Then the proteins were

ensembled

using the

mGluR

(PDB code: 2E4U) as a template with VMD

Finally 2 new models for the

transmembrane

region were performed. (Not enough knowledge to get reliable models)

T1RS MODELING

Slide95

T1RS MODELING (

Evaluation

)Prosa

veredict

:

t1r2

t1r3

Template

(2E4U)

Slide96

Superimposition with template:

T1RS MODELING (

Evaluation

)

Slide97

Superimposition with templates:

T1RS MODELING (

Evaluation

)

Slide98

General Structure:

VFT Domain: A 500 residues with two open twisted

α/β

. With an open cavity where the binding pocket is.

T1RS MODELING

Slide99

Binding

pocket

Open

twisted

α

/

β

Slide100

Polar

residues

Charged

residues

Slide101

General Structure:

VFT Domain: A 500 residues with two open twisted

α/β

.

With an open cavity where the binding pocket is.

CRD: 70 residues long region with 6 paired beta sheets. 5 disulfide bonds between the conserved

Cysteins

.

T1RS MODELING

Slide102

Disulfide

Bonds in

the CRD

Superimposed

with

2E4U

(

mGluR

)

Slide103

PHE

ALA

Disulfide

Bonds

Disulfide

Bonds

SEEMS TO BE IMPORTANT IN THE RECOGNITION OF THE BRAZZEIN

T1R3 CRD

Slide104

General Structure:

VFT Domain: A 500 residues with two open twisted

α/β

.

With an open cavity where the binding pocket is.

CRD: 70 residues long region with 6 paired beta sheets. 5 disulfide bonds between the conserved

Cysteins

.

TMD: 300 residues in the typical 7TM Domain

. Interaction with

lactisole

and cyclamate in this domain.

T1RS MODELING

Slide105

Poorly

modeled

Slide106

VTF

Domain

CRD

Transmembrane

Domain

Slide107

Conclusions

Relative good extracellular model

(goodhomology between class C GPCR)

Bad model in the

transmembrane

domain. Not as good homology and very hard to model a TMD.

Poorly studied binding pockets experimentally, all three domains are related to different

ligands

.

A lot of work to do in refining yet.

New family, lacks annotation in a lot of species (we guess)

T1RS MODELING

Slide108

THANK YOU

!

QUESTIONS?