g protein coupled receptors
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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
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
Comparison of amino acid sequences of these receptors reveal modest conservation ranging from 22% to 64% sequence identity
CLASS A - STRUCTURAL ANALYSIS
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%
CLASS A - STRUCTURAL ANALYSIS
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…)
CLASS A - STRUCTURAL ANALYSIS
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:
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
WXPF/Y motif in TMH6
CLASS A - STRUCTURAL ANALYSIS
MSA of Transmembrane Helix VI (TMH6) of all 5
receptors
NPXIY motif in TMH7
CLASS A - STRUCTURAL ANALYSIS
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
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
CLASS A - STRUCTURAL ANALYSIS
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
CLASS A - STRUCTURAL ANALYSIS
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)
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 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
CLASS A - STRUCTURAL ANALYSIS
CYS 190 (ECL2)
CYS 184 (ECL2)
CYS 106 (TMH3)
CYS 191 (ECL2)
CLASS A - STRUCTURAL ANALYSIS
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?
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
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)
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
DISULFIDE BRIDGES
PHE 168
GLU 169RANDOM COIL (ECL2)
CLASS A - STRUCTURAL ANALYSIS
DISULFIDE BRIDGE
PHE 168
GLU 169
RANDOM COIL (ECL2)
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
11-CIS-RETINAL
W265 (Toggle switch)
CLASS A - STRUCTURAL ANALYSIS
11-CIS-RETINAL
CLASS A - STRUCTURAL ANALYSIS
CLASS A - STRUCTURAL ANALYSIS
TRP265
LYS 296
PHE 261
PHE 212
MET207TYR191
GLU 181
GLU 113
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
LYS 296
11-CIS-RETINAL
TRP265
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
CLASS A - STRUCTURAL ANALYSISBINDING POCKET
GLU134
THR251
GLU 247
IONIC LOCK
ARG135
TMH6
TMH3
CLASS A - STRUCTURAL ANALYSIS
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
CLASS A - STRUCTURAL ANALYSIS
CARAZOLOL
W286 (Toggle switch)
CLASS A - STRUCTURAL ANALYSIS
CLASS A - STRUCTURAL ANALYSIS
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)
β-adrenergic ligands interact with the receptor through two cluster of polar interactions:
CLASS A - STRUCTURAL ANALYSIS
SER203
ASN312 SER207
SER204
TYR316
CLASS A - STRUCTURAL ANALYSIS
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
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
ASN312
CLUSTER OF SERINES
ASN113
TYR316
CLASS A - STRUCTURAL ANALYSIS
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
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 ANALYSIS
SER203
SER207
SER204
TRP286
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
ZM241385
W246(Toggle switch)
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
TRP246
TMH5
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 THM5But ECL2 also plays an important role in binding affinity, through interacting with Glu169 and Phe168
CLASS A - STRUCTURAL ANALYSIS
PHE168
GLU169
ECL2
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
TYR112
ASN102
ASN101
DRY
TYR103
ICL2
ADENOSINE RECEPTOR
CLASS A - STRUCTURAL ANALYSIS
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 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:
T1RS MODELING (Evaluation)
Superimposition with templates:
T1RS MODELING (Evaluation)
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
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
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
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
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?
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