1. Computational and Systems Biology
  2. Structural Biology and Molecular Biophysics
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Common activation mechanism of class A GPCRs

  1. Qingtong Zhou
  2. Dehua Yang
  3. Meng Wu
  4. Yu Guo
  5. Wanjing Guo
  6. Li Zhong
  7. Xiaoqing Cai
  8. Antao Dai
  9. Wonjo Jang
  10. Eugene I Shakhnovich
  11. Zhi-Jie Liu
  12. Raymond C Stevens
  13. Nevin A Lambert
  14. M Madan Babu  Is a corresponding author
  15. Ming-Wei Wang  Is a corresponding author
  16. Suwen Zhao  Is a corresponding author
  1. ShanghaiTech University, China
  2. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China
  3. University of Chinese Academy of Sciences, China
  4. Augusta University, United States
  5. Harvard University, United States
  6. MRC Laboratory of Molecular Biology, United Kingdom
  7. Fudan University, China
Research Article
Cite this article as: eLife 2019;8:e50279 doi: 10.7554/eLife.50279
9 figures, 5 tables and 2 additional files

Figures

Figure 1 with 1 supplement
An increasing number of reported class A GPCR structures facilitates studies on common activation mechanism.

(a) Distribution of structures in different states in the non-olfactory class A GPCR tree as of October 1, 2018. (b) Common GPCR activation mechanism and the residue-level triggers are not well understood.

Figure 1—source data 1

The released class A GPCR structures (as of October 1, 2018).

https://cdn.elifesciences.org/articles/50279/elife-50279-fig1-data1-v3.xlsx
Figure 1—source data 2

Disease mutations occurred in class A GPCRs.

https://cdn.elifesciences.org/articles/50279/elife-50279-fig1-data2-v3.xlsx
Figure 1—figure supplement 1
The pattern of conservation of residues and the map of number of disease-associated mutations in human class A GPCRs.

The alignment of 286 non-olfactory, class A human GPCRs were obtained from the GPCRdb (Hauser et al., 2018; Isberg et al., 2016; Pándy-Szekeres et al., 2018; Isberg et al., 2015) and sent for the sequence conservation score calculation for all residue positions by the Protein Residue Conservation Prediction (Capra and Singh, 2007) tool with scoring method ‘Property Entropy” (Mirny and Shakhnovich, 1999). To obtain disease-associated mutations, we performed database integration and literature investigation for all 286 non-olfactory class A GPCRs. Four commonly used databases (UniProt [The UniProt Consortium, 2017], OMIM [Amberger et al., 2011], Ensembl [Zerbino et al., 2018] and GPCRdb) were first filtered by disease mutations and then merged. Finally, we collected 435 disease mutations from 61 class A GPCRs (Figure 1—source data 2).

Figure 2 with 1 supplement
Understanding GPCR activation mechanism by RRCS and ∆RRCS.

(a) Comparison of residue contact (RC) (Venkatakrishnan et al., 2016) and residue residue contact score (RRCS) calculations. RRCS can describe the strength of residue-residue contact quantitatively in a much more accurate manner than the Boolean descriptor RC. (b) RRCS and ΔRRCS calculation for a pair of active and inactive structures can capture receptor conformational change upon activation. (c) Two types of conformational changes (i.e. switching and repacking contacts) can be defined by RRCS to quantify the global, local, major and subtle conformational changes in a systematic way. (d) Two criteria of identifying conserved residue rearrangements upon receptor activation by RRCS and ΔRRCS. Thirty-four residues pairs were identified based on the criteria (see Materials and methods, Figure 2—source datas 1 and 2 for details), only six of them were discovered before (Venkatakrishnan et al., 2016).

Figure 2—source data 1

Calculated RRCS of 34 residue pairs constituting the common activation pathway for released class A GPCR structures.

https://cdn.elifesciences.org/articles/50279/elife-50279-fig2-data1-v3.xlsx
Figure 2—source data 2

Thirty-four residue pairs show conserved rearrangements of residue contacts upon activation.

https://cdn.elifesciences.org/articles/50279/elife-50279-fig2-data2-v3.pdf
Figure 2—figure supplement 1
Calculation of RRCS and ΔRRCS.

(a) Workflow of RRCS calculation. (b) Examples of RRCS and ΔRRCS calculation for two residues pairs. (c) Statistics of residue contacts and contact types for six receptors (bRho, β2AR, M2R, µOR, A2AR and κ-OR) in their inactive and active states. Contact type describes physicochemical properties of two interacted amino acids that form a pair. The amino acids with hydrophobic side chains (one-letter code: A, V, I, L, M, P, F, Y, W) contribute to the majority of residue contacts, either within themselves (50.1%) or with other amino acids (37.8%).

Figure 3 with 1 supplement
Common activation pathway of class A GPCRs.

Node represents structurally equivalent residue with the GPCRdb numbering (Isberg et al., 2016) while the width of edge is proportional to the average ∆RRCS among six receptors (bRho, β2AR, M2R, µOR, A2AR and κ-OR). Four layers were qualitatively defined based on the topology of the pathway and their roles in activation: signal initiation (layer 1), signal propagation (layer 2), microswitches rewiring (layer 3) and G-protein coupling (layer 4).

Figure 3—figure supplement 1
Rearrangements of ligand-residue contacts in ligand-binding pocket are not conserved, reflecting diverse ligand recognition modes.

(a) Sphere representation of antagonist- and agonist-bound receptor crystal structures. (b) Diverse LRCS and ∆LRCS reveal the repertoire of ligand recognition across class A GPCRs. The agonist or antagonist was treated as a single residue when calculating LRCS and ∆LRCS. As shown by the calculated ∆RRCS, no ligand-residue pair exhibits conserved rearrangements upon activation. (c) Conserved conformational changes were only observed at the very bottom of ligand-binding pocket (6×48, 3×40 and 6×44).

Figure 4 with 2 supplements
The common activation mechanism is the shared portion of various downstream pathways of different class A GPCRs.

(a) Intracellular binding partners used in the active state structures. (b) Comparison of RRCS for active (green) and inactive (orange) states of eight receptors with different intracellular binding partners, including four recently solved cryo-EM structures of Gi/o-bound receptors (5-HT1B receptor, rhodopsin, A1R and µOR) (Tsai et al., 2018; García-Nafría et al., 2018; Kang et al., 2018; Koehl et al., 2018; Draper-Joyce et al., 2018) whose resolutions were low (usually ≥3.8 Å for the GPCR part). Nevertheless, almost all conserved residue rearrangements in the pathway can be observed from them. Three of 34 residues pairs were shown here, see Figure 4—figure supplements 1 and 2 for the remaining 31 residue pairs.

Figure 4—figure supplement 1
The switching conformation change is conserved upon receptor activation.

Comparison of RRCS for active (green) and inactive (orange) states of eight receptors with different intracellular binding partners, including four recently solved cryo-EM structures of Gi/o-bound receptors (5-HT1B receptor, rhodopsin, A1R and µOR) whose resolutions were low (usually ≥3.8 Å for the GPCR part). Nevertheless, almost all conserved residue rearrangements in the pathway can be observed from them. Nineteen of 34 residues pairs were shown here, see Figure 4 and Figure 4—figure supplement 2 for the remaining residue pairs.

Figure 4—figure supplement 2
The repacking conformation change is conserved upon receptor activation.

Comparison of RRCS for active (green) and inactive (orange) states of eight receptors with different intracellular binding partners, including four recently solved cryo-EM structures of Gi/o-bound receptors (5-HT1B receptor, rhodopsin, A1R and µOR) whose resolutions were low (usually ≥3.8 Å for the GPCR part). Nevertheless, almost all conserved residue rearrangements in the pathway can be observed from them. Twelve of 34 residues pairs were shown here, see Figure 4 and Figure 4—figure supplement 1 for the remaining residue pairs.

Figure 5 with 2 supplements
Common activation model of class A GPCRs reveals major changes upon GPCR activation.

(a) Active and inactive state structures form compact clusters in the 2D inter-helical contact space: RRCSTM3-TM7 (X-axis) and RRCSTM3-TM6 (Y-axis). GPCR activation is best described by the outward movement of TM6 and inward movement of TM7, resulting in switch in the contacts of TM3 from TM6 to TM7. (b) Common activation model for class A GPCRs. Residues are shown in circles, conserved contact rearrangements of residue pairs upon activation are denoted by lines.

Figure 5—figure supplement 1
Global conformational change upon activation.

(a) Distinct clustering of inactive- and active-state structures in two-dimentional inter-helical contact space RRCSTM5-TM6 vs. RRCSTM3-TM6. (b) The inter-helical contacts comparison between inactive- and active-state structures. (c) Receptor-specific inter-helical contacts for all class A GPCR structures (inactive, intermediate and active states are colored in orange, cyan and green, respectively). These results demonstrate that receptor activation involves the elimination of TM3-TM6 contacts, formation of TM3-TM7 and TM5-TM6 contacts, reflecting the outward movement of the cytoplasmic end of TM6 away from TM3, the inward movement of TM7 towards TM3 and the repacking of TM5 and TM6.

Figure 5—figure supplement 2
An inverse-agonism of class A GPCRs by preventing the collapse of Na+ pocket.

(a) Comparison of binding poses of antagonist (bosentan [Shihoya et al., 2017]) and inverse agonists (IRL2500 [Nagiri et al., 2019], BIIL260 [Hori et al., 2018] and ritanserin [Peng et al., 2018]). Inverse agonists diffuse deeper in the ligand-binding pocket to touch the Na+ pocket. These key residues around the Na+ pocket are shown. (b) Comparison of the sum of contact scores of the conserved residue pairs around the Na+ pocket (RRCSsodium_pocket) between inactive- and active-state structures. The collapse of the Na+ pocket leads to a denser repacking of six residues (five residue pairs), reflected by higher RRCSsodium_pocket scores compared to that of the inactive state structures. (c) Distribution of three inverse agonist bound structures in the 2D inter-helical contact space: RRCSTM3-TM7 (X-axis) and RRCSTM3-TM6 (Y-axis). These inverse agonists bound structures (5×33, 6BQG and 6K1Q) located in the inactive state region with zero RRCSTM3-TM7 but high RRCSTM3-TM6 scores, despite deeper binding modes.

Figure 6 with 2 supplements
Experimental validation of the common activation mechanism.

(a) cAMP accumulation assay and (b) radioligand binding assay: both validated the common activation pathway-guided design of CAMs/CIMs for A2AR. Wildtype (WT), CAMs and CIMs are shown in black, green and orange, respectively. (c) Mechanistic interpretation of common activation pathway-guided CAMs/CIMs design. N.D.: basal activity was too high to determine an accurate EC50 value.

Figure 6—source data 1

Functional and ligand binding properties of A2AR mutations.

https://cdn.elifesciences.org/articles/50279/elife-50279-fig6-data1-v3.docx
Figure 6—source data 2

Analysis of the 14 unsuccessful predictions of A2AR CAMs/CIMs.

ΔStability (>0 means destabilized;<0 means stabilized) is the change of receptor stability when a mutation was introduced, calculated by Residue Scanning module in BioLuminate (Beard et al., 2013). WT, wild-type.

https://cdn.elifesciences.org/articles/50279/elife-50279-fig6-data2-v3.docx
Figure 6—figure supplement 1
Experimental validation of common activation pathway-guided CAM/CIM design for A 2A R.

(a) Cell surface expression of the WT A 2AR and its mutants. WT, CAMs and CIMs are colored by black, orange and green, respectively. (b) Mapping of validated CAMs/CIMs to the common activation pathway. (c) The mechanisms of CAM/CIM design. CAMs and CIMs are in green and orange, respectively.

Figure 6—figure supplement 2
Experimental validation of common activation pathway-guided CAM/CIM design for Gs-coupled 5-HT7 and Gi-coupled 5-HT1B receptors.

(a) cAMP assay of Gs-coupled 5-HT7 receptor used an EPAC-based BRET cAMP sensor (n = 6). WT, CAMs and CIMs are colored by black, orange and green, respectively. (b) Trafficking assay measured WT and mutant 5-HT7 receptors in the plasma membrane (PM). (c) cAMP measurement of 5-HT1B receptor. CAMs and CIMs are shown in green and orange, respectively. (d) Cell surface expression of mutant 5-HT1B receptors relative to that of WT. DHE, dihydroergotamine.

Figure 7 with 2 supplements
Importance of the common activation pathway in pathophysiological and biological contexts.

(a) Comparison of disease-associated mutations in the common activation pathway (further decomposed into layers 1–4), ligand-binding pocket, G-protein-coupling region and other regions. Red line denotes the mean value. (b) Mapping of disease-associated mutations in class A GPCRs to the common activation pathway. (c) Key roles of the residues constituting the common activation pathway have been reported in numerous experimental studies on class A GPCRs. Two hundred seventy two (272) CAMs/CIMs from 41 receptors were mined from the literature for the 14 hub residues (i.e. residues that have more than one edges in the pathway).

Figure 7—source data 1

Constitutively activating/inactivating mutations for the 14 hub residues in the common activation pathway.

https://cdn.elifesciences.org/articles/50279/elife-50279-fig7-data1-v3.xlsx
Figure 7—figure supplement 1
The common activation pathway can be used to mechanistically interpret disease-associated mutations and CAMs/CIMs.

(a) Pathway-guided mechanistic interpretations of two disease mutations. (b) Pathway-guided mechanistic interpretations of four CAMs/CIMs.

Figure 7—figure supplement 2
Residues in the common activation pathway are more conserved than other functional regions of GPCR.

(a) Illustration of different functional regions of GPCR. (b-d) Sequence pattern of the G protein-coupling region (b), ligand-binding pocket (c) and the common activation pathway (d). (e) Distribution of sequence identity (left) and similarity (right) for functional regions across 286 non-olfactory class A receptors.

Author response image 1
Receptor activation process via intermediate structures (i.e., only agonist but not G protein bound structures) are in receptor- and ligand-specific manner, thereby highlighting the modular nature and complexity of the activation pathways.
Author response image 2
Calculated RRCS for the HETX motif in class B GPCRs.

Tables

Author response table 1
List of twelve structures of six receptors.
GPCRPDB codeResolution (Å)Ligand
G protein/G protein memetic
StateMutation in the TM region
bRho1GZM2.711-cis-retinal (inverse agonist, covalently bound)InactiveNo mutation
3PQR2.9All-trans-retinal (agonist, covalently bound)
GαCT2 (G protein peptide)
ActiveNo mutation
β2AR2RH12.4Carazolol (inverse agonist)InactiveNo mutation
3SN63.2BI-167107 (agonist)
Gs (G protein)
ActiveNo mutation
M2R3UON3.03-quinuclidinyl-benzilate (antagonist)InactiveNo mutation
4MQS3.5Iperoxo (agonist)
Nb9-8 (nanobody)
ActiveNo mutation
μOR4DKL2.8β-funaltrexamine (antagonist)InactiveNo mutation
5C1M2.1BU72 (agonist)
Nb39 (nanobody)
ActiveNo mutation
A2AR3EML2.6ZM241385 (antagonist)InactiveNo mutation
5G533.4NECA (agonist)
Mini-Gs (engineered G protein)
ActiveNo mutation
κOR4DJH2.9JDTic (antagonist)InactiveI135L (3×29)
6B733.1MP1104 (agonist)
Nb39 (nanobody)
ActiveI135L (3×29)
Author response table 2
List of GPCR-G protein complex structures.
ReceptorPDB codeMethodResolution (Å)Ligand + G protein/G protein memetic
A2AR5G53X-ray3.4NECA + Mini-Gs
A2AR6GDGcryo-EM4.1NECA + Mini-Gs
β2AR3SN6X-ray3.2BI-167107 + Gs
β2AR6E67X-ray3.7BI-167107 + Fused Gs C-terminal
Rho6CMOcryo-EM4.5Gi
Rho6FUFX-ray3.1all-trans-retinal +Mini-Go
μOR6DDE/6DDFcryo-EM3.5DAMGO + Gi
A1AR6D9Hcryo-EM3.6Adenosine + Gi2
5-HT1B6G79cryo-EM3.8Donitriptan + Go
CB16N4Bcryo-EM3.0MDMB-Fubinaca + Gi
NTSR16OSAcryo-EM3.0JMV449 + Gi1 (NC state)
NTSR16OS9cryo-EM3.0JMV449 + Gi1 (C state)
M2R6OIKcryo-EM3.6Iperoxo + LY2119620 +GoA
M1R6OIJcryo-EM3.3Iperoxo + G11
Author response table 3
List of three inverse agonist-bound structures.
GPCRPDB codeLigandLigand typeNa+ mimic group in ligandOccupying Na+ pocket
BLT15X33BIIL260Inverse agonistYesYes
ETB6K1QIRL2500Inverse agonistNoYes
5-HT2C6BQHRitanserinInverse agonistNoNo
Author response table 4
List of allosteric modulators among determined structures of class A GPCRs.
Location of binding siteReceptorLigandLigand typePDB code
Intracellular pocketβ2ARCMPD-15PANegative allosteric modulator5X7D
CCR9VercirnonAntagonist5LWE
CCR2CCR2-RA-[R]Antagonist5T1A
Extra-helical binding siteC5aRNDT9513727Inverse agonist5O9H
C5aRavacopanAllosteric antagonist6C1R
C5aRNDT9513727Allosteric antagonist6C1Q
β2ARcompound-6FAPositive allosteric modulator6N48
P2Y1BPTUantagonist4XNV
GPR40AP8Full allosteric agonists (agoPAM)5TZY
GPR40Compound 1Full agonist5KW2
PAR2AZ3451Antagonist5NDZ
Non-canonical binding pocketGPR40TAK-875Ago-allosteric modulator4PHU
GPR40MK-8666Ago-allosteric modulator5TZR
PAR2AZ8838antagonist5NDD
Extracellular vestibule above the orthosteric siteM2LY2119620Positive allosteric modulator4MQT
Author response table 5
List of available class B GPCRs structures.
ReceptorPDB codeMethodResolution (Å)Ligand + G protein/G protein memeticStates
CRF1R4K5YX-ray2.9Antagonist CP-376395Inactive
CRF1R4Z9GX-ray3.2Antagonist CP-376395Inactive
CTR6NIYcryo-EM3.3Peptide ligand + GsActive
CTR5UZ7cryo-EM4.1Peptide ligand + GsActive
CGRP6E3Ycryo-EM3.3CGRP + GsActive
GCGR4L6RX-ray3.3No ligand was seenInactive
GCGR5EE7X-ray2.5Antagonist MK-0893Inactive
GCGR5YQZX-ray3.0Partial agonist NNC1702Intermediate
GCGR5XEZX-ray3.0Negative allosteric modulator NNC0640Inactive
GCGR5XF1X-ray3.2Negative allosteric modulator NNC0640Inactive
GLP-1R5VAIcryo-EM4.1human GLP-1 + GsActive
GLP-1R6B3Jcryo-EM3.3Exendin-P5 + GsActive
GLP-1R5NX2X-ray3.7Truncated peptide agonistIntermediate
GLP-1R5VEWX-ray2.7PF-06372222Inactive
GLP-1R5VEXX-ray3.0NNC0640Inactive
PTH1R6FJ3X-ray2.5Peptide agonist ePTHIntermediate
PTH1R6NBHcryo-EM3.5A long-acting PTH analog + GsActive
PTH1R6NBIcryo-EM4.0A long-acting PTH analog + GsActive
PTH1R6NBFcryo-EM3.0A long-acting PTH analog + GsActive

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 6 and 7.

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