g protein coupled receptors

G PROTEIN COUPLED RECEPTORS

1. GPCR FAMILY

2. CLASS A STRUCTURAL ANALYSIS

3. TASTE RECEPTORS

4. CONCLUSIONS & QUESTIONS

GPCRS. OVERVIEW

Also known as 7TM receptorsLargest 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

Membrane proteins with seven transmembrane domains

Upon activation, signal gets transmitted to the cytoplasmatic face and amplifies through heterotrimeric G protein complex

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

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)

GPCRs CLASS A - STRUCTURAL ANALYSIS

1. CLASS A FAMILY OVERVIEW

2. SEQUENCE SIMILARITIES. CONSERVED MOTIFS

3. STRUCTURAL ANALYSIS• EXTRACELLULAR REGION• LIGAND BINDING POCKET (TRANSMEMBRANE)• INTRACELLULAR REGION

4. CONCLUSIONS & QUESTIONS

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 ECL2

Some 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

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


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

Comparison of amino acid sequences of these receptors reveal modest conservation ranging from 22% to 64% sequence identity


SQUIDRHODOPSIN

BOVINERHODOPSIN

ADENOSINE 2A RECEPTOR

β-1 ADREN. RECEPTOR

β-2 ADREN.RECEPTOR

SQUID RHODOPSIN 27% 22% 25% 25%

BOVINERHODOPSIN 27% 22% 24% 23%

ADENOSINE2A

RECEPTOR22% 22% 36% 33%

β-1 ADREN. RECEPTOR 25% 24% 36% 64%

β-2 ADREN. RECEPTOR 25% 23% 33% 64%


Percentage of sequence identity within receptors

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…)


MSA of the firs Transmembrane Helix I

(TMH1) of all 5 receptors


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

Conserved segments are localized in the transmembrane domains, among them the most highly conserved are:

E/DRY motif in TMH3


MSA of Transmembrane Helix III (TMH3) of all 5

receptors

WXPF/Y motif in TMH6


MSA of Transmembrane Helix VI (TMH6) of all 5

receptors

NPXIY motif in TMH7


MSA of Helix VII (TMH7) of all 5 receptors

CLASS A - STRUCTURAL ANALYSISβ-2 ADRENERGIC

RECEPTOR

RHODOPSIN (Bovine)

ADENOSINE-2A RECEPTOR)

RHODOPSIN (Squid)

β-1 ADRENERGIC RECEPTOR


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


EXTRACELLULAR REGION

RHODOPSINExtensive 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

CLASS A - STRUCTURAL ANALYSISN-TERMINUS

ECL-2

ECL-1

ECL-3


EXTRACELLULAR REGION

RHODOPSINExtensive 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 restrictedOne disulfide bridge (it has been shown to be essential

for the normal function of Rhodopsin)


CYS 187 (ECL2)

CYS 110 (TMH3)


Β-ADRENERGIC RECEPTORS

Extracellular region much more openShort 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 TMH3The random coil section of ECL2 forms the top of the

ligand binding pocket (only partially occluded)ECL3 forms no interaction with ECL1 or ECL2


CYS 190 (ECL2)

CYS 184 (ECL2)

CYS 106 (TMH3)

CYS 191 (ECL2)


Β-ADRENERGICExtracellular region much more openShort 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 TMH3The random coil section of ECL2 forms the top of the

ligand binding pocket (only partially occluded)ECL3 forms no interaction with ECL1 or ECL2Entire 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?


RHODOPSIN Β-ADRENERGIC RECEPTOR

?


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


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)


ADENOSIN RECEPTORS



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


DISULFIDE BRIDGES

PHE 168

GLU 169RANDOM COIL (ECL2)


DISULFIDE BRIDGE

PHE 168

GLU 169

RANDOM COIL (ECL2)


ADENOSIN RECEPTORS



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


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.



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

11-CIS-RETINAL

W265 (Toggle switch)


11-CIS-RETINAL


TRP265

LYS 296

PHE 261

PHE 212

MET207TYR191

GLU 181

GLU 113



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


LYS 296

11-CIS-RETINAL

TRP265



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

CLASS A - STRUCTURAL ANALYSISBINDING POCKET

GLU134

THR251

GLU 247

IONIC LOCK

ARG135

TMH6

TMH3


β-ADRENERGIC RECEPTORSSimilar 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


CARAZOLOL

W286 (Toggle switch)



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:


SER203

ASN312 SER207

SER204

TYR316



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

β-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


ASN312

CLUSTER OF SERINES

ASN113

TYR316



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

β-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 TMH7• The second group comprises a cluster of serine residues on TMH5


SER203

SER207

SER204

TRP286


ADENOSIN 2A

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


ZM241385

W246(Toggle switch)


ADENOSINE 2A


This pocket changes in position and orientation with respect to both rhodopsin and adrenergic receptors


TRP246


ADENOSINE 2A



Adenosin ligand ZM241385 forms mainly polar interactions with THM5


TRP246

TMH5


ADENOSINE 2A



Adenosin ligand ZM241385 forms mainly polar interactions with THM5But ECL2 also plays an important role in binding affinity, through interacting with Glu169 and Phe168


PHE168

GLU169

ECL2


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


TYR112

ASN102

ASN101

DRY

TYR103

ICL2

ADENOSINE RECEPTOR


CONCLUSIONSExtracellular and intracellular regions show

more diversity Conserved disulfide bridges stabilise

extracellular domainTransmembrane region is more structurally

conservedTRP acts as toogle switch rotamer and is

conserved in all structures solved to dateIonic lock theory just valid for RhodopsinDRY motif conserved throughout but

functions remain still not fully knwon

CASE STUDY:TASTE RECEPTORS

1. TASTE RECEPTORS OVERVIEW

2. CONSERVATION

3. MODELING

4. STRUCTURE

5. CONCLUSIONS

Five basic tastes:Salty SourBitter UmamiSweet

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

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

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

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 sucralose

Antagonists: Lactisole.

SWEET RECEPTOR (T1RS/T1R3) LIGANDS

T1RS RECEPTORS

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.

BITTER

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

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

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

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

T1Rs SignalThe 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

T1Rs Venus Flytrap (VFT)Good general conservation.Loop regions with more variability.

T1RS CONSERVATION

T1Rs Venus Flytrap (VFT)

T1RS CONSERVATION

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

T1Rs Tansmembrane Domain:

T1RS CONSERVATION

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

T1RS CONSERVATION

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

T1RS MODELING

MODELING (With modeller)

ALIGNMENT REFINEMENT (Cysteins residues)

HMM BUILDING AND ALIGNING

STRUCTURAL ALIGNMENT (STAMP)

SEQUENCES RETRIEVAL

Psi-BLAST 3 Different glutamate receptors and a peptide receptor

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 VMDFinally 2 new models for the transmembrane

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

T1RS MODELING

T1RS MODELING (Evaluation)

• Prosa veredict:

t1r2 t1r3

Template (2E4U)

Superimposition with template:


Superimposition with templates:


General Structure:VFT Domain: A 500 residues with two open

twisted α/β. With an open cavity where the binding pocket is.

T1RS MODELING

Binding pocket

Open twisted α/β

Polar residues

Charged residues



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

T1RS MODELING

Disulfide Bonds in the CRDSuperimposed with 2E4U(mGluR)

PHE

ALA

Disulfide BondsDisulfide Bonds

SEEMS TO BE IMPORTANT IN THE RECOGNITION OF THE BRAZZEIN

T1R3 CRD



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

Poorly modeled

VTF Domain

CRD

TransmembraneDomain

ConclusionsRelative 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

THANK YOU!QUESTIONS?

g protein coupled receptors

Documents

structural analysisclass

structural analysispdbs

structural analysisgpcrs

small class

gpcrsgpcrs class

rhodopsin r class

sequence identity class

structural analysis