G PROTEIN COUPLED
RECEPTORS
1. GPCR FAMILY
2. CLASS A STRUCTURAL ANALYSIS
3. TASTE RECEPTORS
4. CONCLUSIONS & QUESTIONS
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
 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
CLASS A - STRUCTURAL ANALYSIS
 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
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
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
CLASS A - STRUCTURAL ANALYSIS
 Comparison of amino acid sequences of these
receptors reveal modest conservation ranging from
22% to 64% sequence identity
CLASS A - STRUCTURAL ANALYSIS
Percentage of sequence identity within receptors
SQUID
RHODOPSIN
SQUID
RHODOPSIN
BOVINE
RHODOPSIN
ADENOSINE
2A RECEPTOR
β-1 ADREN.
RECEPTOR
β-2 ADREN.
RECEPTOR
27%
22%
25%
25%
22%
24%
23%
36%
33%
BOVINE
RHODOPSIN
27%
ADENOSINE
2A
RECEPTOR
22%
22%
β-1 ADREN.
RECEPTOR
25%
24%
36%
β-2 ADREN.
RECEPTOR
25%
23%
33%
64%
64%
CLASS A - STRUCTURAL ANALYSIS
 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
CLASS A - STRUCTURAL ANALYSIS
MSA of the five receptors structurally solved identified
25 conserved residues:
CLASS A - STRUCTURAL ANALYSIS
 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
CLASS A - STRUCTURAL ANALYSIS
 WXPF/Y motif in TMH6
MSA of Transmembrane
Helix VI (TMH6) of all 5
receptors
CLASS A - STRUCTURAL ANALYSIS
 NPXIY motif in TMH7
MSA of Helix VII (TMH7) of
all 5 receptors
ADENOSINE-2A
RECEPTOR)
RHODOPSIN
(Squid)
CLASS A - STRUCTURAL ANALYSIS
β-1 ADRENERGIC
RECEPTOR
β-2 ADRENERGIC
RECEPTOR
RHODOPSIN
(Bovine)
CLASS A - STRUCTURAL ANALYSIS
Structural superpositioning
of the 5 receptors
demonstrating a high
level of overall structure
similarity
RMSDs of superimposition
ranging from 0.63Å to
4.03Å
Slightly more variation at
the extracellular side of
the membrane surface
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 βsheet with additional interactions ECL3-ECL1
Access to retinal binding pocket severely restricted
CLASS A - STRUCTURAL ANALYSIS
N-TERMINUS
ECL-1
ECL-2
ECL-3
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 βsheet 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)
CLASS A - STRUCTURAL ANALYSIS
CYS 187 (ECL2)
CYS 110 (TMH3)
CLASS A - STRUCTURAL ANALYSIS
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
CLASS A - STRUCTURAL ANALYSIS
CYS 184 (ECL2)
CYS 106 (TMH3)
CYS 190 (ECL2)
CYS 191 (ECL2)
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?
CLASS A - STRUCTURAL ANALYSIS
?
RHODOPSIN
Β-ADRENERGIC RECEPTOR
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
CYS 259
CLASS A - STRUCTURAL ANALYSIS
(ECL3)
CYS 262
(TMH6)
CYS 71(ECL1)
CYS 166 (ECL2)
CYS 77 (TMH3)
CYS 159 (ECL2)
CYS 74
(TMH3)
CYS 146 (NTERMINUS)
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
CLASS A - STRUCTURAL ANALYSIS
PHE 168
DISULFIDE BRIDGES
GLU 169
RANDOM COIL (ECL2)
RANDOM COIL (ECL2)
CLASS A - STRUCTURAL
ANALYSIS
DISULFIDE BRIDGE
GLU 169
PHE 168
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
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.
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
W265 (Toggle switch)
11-CIS-RETINAL
CLASS A - STRUCTURAL ANALYSIS
11-CIS-RETINAL
CLASS A - STRUCTURAL ANALYSIS
CLASS A - STRUCTURAL ANALYSIS
TYR191
MET207
GLU 181
GLU 113
PHE 212
LYS 296
TRP265
PHE 261
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
CLASS A - STRUCTURAL ANALYSIS
11-CIS-RETINAL
TRP265
LYS 296
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
BINDING
POCKET
CLASS A - STRUCTURAL ANALYSIS
TMH6
THR251
ARG135
TMH3
GLU134
IONIC LOCK
GLU 247
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
CLASS A - STRUCTURAL ANALYSIS
W286 (Toggle switch)
CARAZOLOL
CLASS A - STRUCTURAL ANALYSIS
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:
CLASS A - STRUCTURAL ANALYSIS
SER204
TYR316
SER203
ASN312
SER207
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
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
CLASS A - STRUCTURAL ANALYSIS
ASN113
CLUSTER OF SERINES
ASN312
TYR316
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
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
CLASS A - STRUCTURAL ANALYSISSER204
SER203
TRP286
SER207
CLASS A - STRUCTURAL ANALYSIS
 ADENOSIN 2A
With the recent elucidation of this structure
(2008), we see a very different location of the
binding pocket
CLASS A - STRUCTURAL ANALYSIS
W246(Toggle switch)
ZM241385
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
CLASS A - STRUCTURAL ANALYSIS
CLASS A - STRUCTURAL ANALYSIS
TRP246
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
CLASS A - STRUCTURAL ANALYSIS
TMH5
TRP246
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
CLASS A - STRUCTURAL ANALYSIS
ECL2
GLU169
PHE168
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
CLASS A - STRUCTURAL ANALYSIS
DRY
ASN101
ADENOSINE
RECEPTOR
ASN102
TYR112
TYR103
ICL2
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 Rhodopsin
DRY 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
TASTE RECEPTORS
Five basic tastes:
Salty
Sour
Bitter
Umami
Sweet
Ligand-gated
cation channels
•G protein-coupled
receptors
•The most important for
food acceptance
Sweet and Umami related with appetitive
sensations
Bitter sense related to the rejection of food
TASTE RECEPTORS
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.
SWEET AND UMAMI
 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 RECEPTOR (T1RS/T1R3) LIGANDS
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.
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 Nterminal extracellular domain but may act as
dimers as well.
BITTER
T1RS CONSERVATION
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 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
T1Rs Venus Flytrap (VFT)
Good general conservation.
Loop regions with more variability.
T1RS CONSERVATION
T1Rs Venus Flytrap (VFT)
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 MODELING
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
SEQUENCES RETRIEVAL
Psi-BLAST
3 Different glutamate receptors and a peptide receptor
STRUCTURAL ALIGNMENT (STAMP)
HMM BUILDING AND ALIGNING
ALIGNMENT REFINEMENT (Cysteins residues)
MODELING (With modeller)
T1RS MODELING
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 (Evaluation)
t1r2
t1r3
• Prosa veredict:
Template (2E4U)
T1RS MODELING (Evaluation)
Superimposition with template:
T1RS MODELING (Evaluation)
Superimposition with templates:
T1RS MODELING
General Structure:
VFT Domain: A 500 residues with two open
twisted α/β. With an open cavity where the
binding pocket is.
Open twisted α/β
Binding pocket
Polar residues
Charged residues
T1RS MODELING
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.
Disulfide Bonds in the CRD
Superimposed with 2E4U
(mGluR)
T1R3 CRD
Disulfide Bonds
Disulfide Bonds
ALA
SEEMS TO BE IMPORTANT
IN THE RECOGNITION OF
THE BRAZZEIN
PHE
T1RS MODELING
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.
Poorly modeled
VTF Domain
CRD
Transmembrane
Domain
T1RS MODELING
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)
THANK YOU!
QUESTIONS?