Analysis of β2AR-Gs and β2AR-Gi complex formation byNMR spectroscopyXiuyan Maa, Yunfei Hub,c

, Hossein Batebid, Jie Henga, Jun Xua, Xiangyu Liua,e, Xiaogang Niub, Hongwei Lib,Peter W. Hildebrandd,f,g, Changwen Jinb,1, and Brian K. Kobilkaa,h,1

aBeijing Advanced Innovation Center for Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China; bBeijing Nuclear MagneticResonance Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100084, China; cInnovation Academy for PrecisionMeasurement Science and Technology, Chinese Academy of Science, Wuhan, 430071, China; dInstitute of Medical Physics and Biophysics, University Leipzig,04107 Leipzig, Germany; eSchool of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China; fInstitute of Medical Physics and Biophysics, CharitéMedical University Berlin, Berlin, Germany; gBerlin Institute of Health, 10178 Berlin, Germany; and hDepartment of Molecular and Cellular Physiology,Stanford University School of Medicine, Stanford, CA 94305

Contributed by Brian K. Kobilka, July 18, 2020 (sent for review May 20, 2020; reviewed by Nevin A. Lambert and Alan Smrcka)

The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimu-latory G protein Gs and stimulates cAMP formation. Functionalstudies have shown that the β2AR also couples to inhibitory Gprotein Gi, activation of which inhibits cAMP formation [R. P. Xiao,Sci. STKE 2001, re15 (2001)]. A crystal structure of the β2AR-Gs

complex revealed the interaction interface of β2AR-Gs and struc-tural changes upon complex formation [S. G. Rasmussen et al.,Nature 477, 549–555 (2011)], yet, the dynamic process of theβ2AR signaling through Gs and its preferential coupling to Gs overGi is still not fully understood. Here, we utilize solution nuclearmagnetic resonance (NMR) spectroscopy and supporting moleculardynamics (MD) simulations to monitor the conformational changesin the G protein coupling interface of the β2AR in response to thefull agonist BI-167107 and Gs and Gi1. These results show thatBI-167107 stabilizes conformational changes in four transmem-brane segments (TM4, TM5, TM6, and TM7) prior to coupling toa G protein, and that the agonist-bound receptor conformation isdifferent from the G protein coupled state. While most of theconformational changes observed in the β2AR are qualitatively thesame for Gs and Gi1, we detected distinct differences between theβ2AR-Gs and the β2AR-Gi1 complex in intracellular loop 2 (ICL2).Interactions with ICL2 are essential for activation of Gs. These dif-ferences between the β2AR-Gs and β2AR-Gi1 complexes in ICL2 maybe key determinants for G protein coupling selectivity.

GPCR | β2-adrenergic receptor | NMR spectroscopy |G protein coupling specificity

Gprotein-coupled receptors (GPCRs) are the largest family ofmembrane proteins responsible for most of the transmem-

brane signal transduction processes. The activation of GPCRs ismediated by diverse stimuli, including neurotransmitters, hor-mones, ions, and photons. It is now well known that GPCRscan activate different signaling pathways through coupling withdistinct downstream transducers, for example G proteins andarrestins (1). It is generally accepted that GPCRs are highlydynamic proteins that can adopt multiple distinct conformationsdepending on the ligands, lipids, environments, and signalingtransducers through a conformational selection mechanism(1, 2). The β2 adrenergic receptor (β2AR) has been an importantmodel system for studying aspects of GPCR biology and phar-macology for more than 40 y. The β2AR can activate more thanone G protein isoform, while preferentially coupling to thestimulatory G protein (Gs), which activates adenylyl cyclase, italso couples to the inhibitory G protein family (Gi/o) (3), whichinhibits adenylyl cyclase (Fig. 1A).The structure of the β2AR-Gs complex revealed the interac-

tion between β2AR and Gs, and the structural changes that takeplace upon complex formation (4), yet, the dynamic processof β2AR signaling through Gs is still not fully understood. Thestructural features of the β2AR-Gs complex have been observed

in GPCR-G protein complexes for Gi, Go, and G11 proteinsdetermined by cryo-electron microscopy (cryo-EM) (5–7); how-ever, the molecular mechanism of G protein coupling specificityis still unclear. This may in part be due to the fact that all of theGPCR-G protein complex structures solved so far are capturedin a biochemically stable nucleotide-free state, while specificitydetermination may happen at an earlier stage of complex for-mation. Single-molecule studies of the β2AR coupling to Gs pro-vide evidence for at least one intermediate state (8). The existenceof intermediate states is further supported by monitoring β2AR-Gscomplex formation using time-resolved radiolytic footprinting andhydrogen-deuterium exchange (9).NMR spectroscopy has previously been used to study GPCR

dynamics (10–16). Several site-specific isotopic labeling strate-gies have been shown to be feasible for studying conformationaldynamics of GPCRs, such as 13C-dimethylated lysine, 13CH3-e-methionine, and 19F-labeled cysteine (17). Previous NMR studiesof the β2AR using these methods have revealed weak allostericcoupling between ligand binding domain and G protein couplingdomain, and that the β2AR can adopt multiple conformationalstates during activation (11–13). However, due to the limitation oflarge molecular weight and difficulty in sample preparation, little is

Significance

Recent structures of GPCRs in complex with G proteins provideimportant insights into G protein activation by family A andfamily B GPCRs; however, important questions remain. Wedon’t fully understand the mechanism of G protein couplingspecificity or coupling promiscuity of some GPCRs. The β2ARpreferentially couples to Gs and less efficiently to Gi, yet β2AR-Gi

coupling has been shown to play important roles in cardiacphysiology. To better understand the structural basis for thepreferential coupling of the β2AR to Gs over Gi, we used NMRspectroscopy and supporting MD simulations to study the con-formational changes in the intracellular surface of the β2AR.These studies reveal a distinct difference in intracellular loop 2interactions with Gs and Gi1.

Author contributions: Y.H., C.J., and B.K.K. designed research; X.M., H.B., X.N., and H.L.performed research; X.M., Y.H., H.B., J.H., J.X., X.L., X.N., H.L., P.W.H., C.J., and B.K.K.analyzed data; and X.M., J.X., X.L., and B.K.K. wrote the paper.

Reviewers: N.A.L., Augusta University; and A.S., University of Michigan Medical School.

Competing interest statement: B.K.K. is co-founder of and consultant for ConfometRx,Inc.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).1To whom correspondence may be addressed. Email: changwen@pku.edu.cn or kobilka@stanford.edu.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2009786117/-/DCSupplemental.

First published August 31, 2020.

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known about the conformational dynamics of the β2AR in re-sponse to different G protein subtypes. In this study, we utilized13C-dimethylated lysine as an NMR probe to investigate the con-formational changes of several sites in the intracellular G protein

binding interface of the β2AR. 13C-dimethylated lysine is an idealprobe for this purpose because of its large, flexible side chain,enabling studies of larger protein complexes. Our studies showthat in addition to TM6, agonist binding can also promote

B

A

C

D

E

Fig. 1. The β2AR-G protein signaling pathways. (A) The agonist-bound β2AR activates either Gs or Gi heterotrimer, which stimulates or inhibits the adenylylcyclase activity, respectively. (B) Structure of β2AR-Gs complex (PDB ID code: 3SN6), the lysine residues that are chosen as NMR probes are shown as solidspheres, other lysine residues were mutated to arginine as described in the text. (C–E) The lysine probes undergo conformational changes during the acti-vation of the β2AR, as shown by cytoplasmic views of active β2AR (PDB ID code: 3SN6) (C), inactive β2AR (PDB ID code: 2RH1) (D), and the overlap of active andinactive β2AR (E).

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conformational changes in TM4, TM5, and TM7. We observefurther structural changes in β2AR coupled to Gs and Gi1. Whilethe structural changes stabilized by Gs and Gi1 are qualitativelysimilar in most cytoplasmic domains probed, we detected distinctdifferences between the β2AR-Gs and the β2AR-Gi1 complex inintracellular loop 2 (ICL2). In the inactive-state structure of theβ2AR, ICL2 is a loop, while in the β2AR-Gs complex, ICL2 formsan α-helix that positions F13934.51 in ICL2 to engage a hydro-phobic pocket in Gαs, thereby triggering nucleotide release (4).

ResultsDesign of Modified β2ARs for 13C-Dimethylated Lysine NMR Studies.13C-dimethylated lysine probes have long served as excellentprobes for NMR experiments due to high sensitivity and rela-tively long transverse relaxation times (18). The WT β2AR con-tains 16 lysine residues, most of which are located at the G proteincoupling interface (Fig. 1 B–E and SI Appendix, Fig. S1A). There-fore, these lysine residues can be used as NMR reporters to monitorstructural changes in response to agonists and G proteins.However, previous studies have shown that the 13C-dimethylated

lysine spectra of C-terminally truncated WT β2AR (β2AR-365N)was too crowded to make assignments (11), probably due to thesimilar chemical environments of these intracellular lysines. Toavoid the NMR signals overlapping, we made a truncated lysine-free β2AR construct (β2AR-365N-zero-K) by mutating all ly-sines to arginines as previously reported (19) and removing theC-terminal 48 residues (SI Appendix, Fig. S1 A and D). Radio-ligand binding studies show that these modifications have onlysmall effects on the affinity of antagonists and agonists (SI Ap-pendix, Fig. S1C). A previous study showed that the lysine-freeβ2AR has functional properties that are comparable to WTβ2AR in stimulation of adenylyl cyclase activity (19) and the re-cruitment of β-arrestin (20). The only reported effect of removinglysine is loss of ubiquitination and subsequent targeting receptorto lysosome for degradation (20). We then individually introducedeight lysines to different transmembrane segments in the intra-cellular domain of the β2AR based on the lysine-free construct(Fig. 1 B–E and SI Appendix, Fig. S1A). These eight residues wereK601.59, K14034.52, K1474.39, K2275.56, K2676.29, K2736.35,R328K7.55, R333K8.51 (superscripts are Ballesteros and Weinsteinnumbering; ref. 21), located at the intracellular end of TM1, TM4,TM5, TM6, TM7, ICL2, and H8. The resulting eight constructswere expressed, purified, and labeled through reductive methyl-ation (SI Appendix, Figs. S1B and S2A). Methylation efficiency washigh, as determined by loss of labeling with fluorescamine, anamine reactive fluorophore (SI Appendix, Fig. S1B). We applied1H-13C heteronuclear single quantum coherence spectroscopy(1H-13C HSQC) to each construct. The signal of each lysine wasfound at around 2.85 ppm in the 1H dimension and 45 ppm in the13C dimension as compared to the spectrum of lysine-free β2AR(SI Appendix, Fig. S3 A–H). The signals are unlikely from the Nterminus because the N-terminal Flag tag was removed aftermethylation. In order to make sure they are not residual N ter-minus signals due to incomplete protein digestion, we measuredthe signals from the methylated N terminus and they are in dif-ferent positions from the lysine signals (SI Appendix, Fig. S3I). Foreach β2AR-365N-one-K construct, we obtained the 1H-13C HSQCspectra under the following conditions: unliganded (apo-state)β2AR, BI-167107-bound, carazolol-bound, BI-167107-bound β2ARin complex with Gs or Gi1 in the presence of 300 μMGDP (Gs

GDP

or Gi1GDP), and BI-167107-bound β2AR in complex with

nucleotide-free Gs or Gi1 (GsEMPTY or Gi1

EMPTY). For thesestudies we used saturating concentrations of BI-167107, a potentfull agonist with a dissociation half-life of 400 min and an asso-ciation half-life of less than 4.4 min (12). For inverse agonist, weused a saturating concentration of carazolol. The very slow dis-sociation half-lives for these ligands ensures that they are boundthroughout the NMR experiment.

There are crystal structures of carazolol-bound β2AR (22) andthe BI-167107–bound β2AR-Gs complex (4); however, it has notbeen possible to crystalize native β2AR in the apo-state or boundto agonist alone, most likely due to the inherent instability of thereceptor under these conditions. While β2AR also couples to Gi,there are no reported structures of a β2AR-Gi complex. There-fore, the NMR experiments provide structural insights into theapo-state, the agonist-bound β2AR, and the β2AR-Gi1 complexrelative to available crystal structures. Figs. 2–4 show the com-parison of 1H-13C HSQC spectra of the eight 13C-dimethylatedlysine probes in TM1, ICL2, TM4, TM5, TM6, TM7, and Helix-8in four different conditions: apo-state, inverse agonist-bound,agonist-bound, and agonist + Gs

EMPTY.

Apo-β2AR Compared to Inverse Agonist-Bound States. The β2ARexhibits basal activity for Gs activation in the apo-state. Thisactivity can be suppressed by inverse agonists such as carazolol.Previous 13C-methionine and 19F NMR studies revealed rela-tively subtle conformational changes when comparing inverse-agonist stabilized inactive-state and the apo-state β2AR (12, 13).Consistent with these findings, our results show relatively smallspectral changes in several domains of the intracellular surface inapo-state and the carazolol-stabilized inactive state. We observethe largest difference between the apo-state and carazolol-boundinactive state is in K601.59 (Fig. 2A), suggesting a change in itschemical environment possibly due to a change in polar interac-tions with the end of Helix 8 (Fig. 2 C–F). In the apo-state, K601.59

is represented by strong Peak 1 with a weak shoulder Peak 2. Incarazolol-bound receptor we observe an up-field shift in Peak 1and no change in Peak 2.In the apo-state we observe two peaks for K1474.39 at the end

of TM4 (Fig. 3), K2736.35 in TM6, and R328K7.55 in TM7(Fig. 4). This suggests the existence of conformational hetero-geneity in these receptor domains with exchange between twostates on slow time scale (seconds or slower). In K2736.35 andR328K7.55 we observe very small up-field shifts in the position ofPeak 1 in carazolol-bound receptor compared to the apo-statethat are in the opposite direction observed for agonist (Fig. 4 Cand D).

Conformational Changes following Agonist Binding and Gs Coupling.To correlate structural changes observed by NMRwith the β2AR-Gscrystal structure, we obtained spectra of the β2AR bound tonucleotide-free Gs (β2AR-Gs

EMPTY). β2AR-GsEMPTY was formed

by adding the nucleotidase apyrase after complex formation todegrade any released GDP. In the β2AR-Gs

EMPTY crystal struc-ture, the largest structural changes are the outward movement ofTM5 and TM6 and inward movement of TM7, which allows theinsertion of the α5 helix of Gαs (Fig. 1 C–E and SI Appendix, Fig.S4). We observed obvious spectral changes when comparing β2ARbound to agonist alone with the β2AR-Gs

EMPTY complex in allprobes except K3338.51 in Helix 8 (Figs. 2–4). This may be due to acombined effect of direct Gs protein binding, as well as a fullyactive conformation stabilized only by agonist and Gs protein, butnot agonist alone (13).ICL1 and helix 8. The spectrum of K601.59 at the end of TM1 isnearly identical for apo-state and agonist-bound state (Fig. 2A),suggesting little perturbation of conformational dynamics in thejunction of TM1 and ICL1. ICL1 and Helix 8 have no directinteraction with Gs in the β2AR-Gs

EMPTY complex. The obser-vation of intensity reduction upon coupling with Gs for K60

1.59

(Fig. 2A) and K3338.51 (Fig. 2B) may result from the slowertumbling due to the formation of complex with a much largermolecular weight. We do observe a change in the spectra of K601.59

upon coupling to Gs. In the inactive state K601.59 forms a polarinteraction with E3388.56 in Helix 8 (Fig. 2 E and F). This is lost inthe β2AR-Gs

EMPTY complex due to a small ∼1.5 Å movement of

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TM1 away from Helix 8 (Fig. 2 C and D). We also observe theappearance of a weak downfield peak in R333K8.51 in H8.ICL2. In the inactive state structure of the β2AR, ICL2 has nosecondary structure while it is an alpha-helix in the β2AR-Gs

EMPTY

complex (Fig. 3 C and D). The helix positions F13934.51 in ICL2 toengage a hydrophobic pocket formed by H41, V217, F376, C379,R380, and I383 in the Gαs subunit (Fig. 3D). This interaction isessential for Gs activation (4, 23, 24). The conformational changesdetected in the NMR spectra of K1474.39 and K14034.52 may rep-resent the chemical environment change during the transition ofICL2 from a loop to a helix (Fig. 3 A and B).K1474.39 is located at the junction of TM4 and ICL2 (Fig. 1 C

and D). In the inactive state, K1474.39 forms a hydrogen bondwith the backbone carbonyl of Q6512.51 in ICL1 (Fig. 3E). Thisinteraction is lost in the structure of β2AR-Gs

EMPTY (Fig. 3F). Inthe apo state, the two peaks likely represent an equilibrium be-tween the formation (Peak 2) and disruption (Peak 1) of thehydrogen bond in slow exchange (Fig. 3B). The observation ofone dominant peak for K1474.39 in the agonist-bound state com-pared to the two peaks in the apo-state most likely suggests fasterexchange rate between the different conformations observed inthe apo state (Fig. 3B). In β2AR-Gs complex we observe pre-dominantly Peak 1, consistent with the loss of the hydrogen bond(Fig. 3B).In contrast to the dynamics in the junction of TM4 and ICL2

observed in the apo and agonist-bound receptor, we observe astrong single peak for K14034.52 at the junction of ICL2 and TM3(Fig. 3A). In the inactive state, K14034.52 forms a hydrogen bond

with Q2295.58 in TM5, while in the β2AR-Gs complex it is adja-cent to the β2AR-Gs interface (Fig. 3 C and D). Coupling with Gsdramatically alters the chemical environment of K14034.52, asevidenced by the appearance of a new downfield peak (labeled aspeak2). Only a small fraction of Peak 1 remains following theformation of β2AR-Gs

EMPTY, consistent with a minor populationof uncoupled β2AR (Fig. 3A).TM5, 6, and 7. While we observe spectral changes in K2275.56,K2676.29, K2736.35, and R328K7.55 upon agonist binding, furtherchanges are observed following G protein coupling (Fig. 4 A–D),consistent with changes observed in TM5, TM6, and TM7 in theβ2AR-Gs structure (Fig. 4 E and F). The slight broadening ofthe peak representing K2275.56 upon agonist binding suggests theappearance of two or more conformations in intermediate ex-change at the intracellular side of TM5 (Fig. 4A). Upon couplingto Gs we see the appearance of a new downfield peak (Peak 2)and a weaker Peak 1 (Fig. 4A).The two TM6 probes, K2676.29 and K2736.35, show significant

differences in terms of peak patterns. In the apo and agonist-bound state K2736.35 is represented by two weak peaks, indicatingthat there are at least two conformations with a slow exchangerate, while K2676.29 is represented by a stronger single peak(Fig. 4 B and C). This is because K2676.29 is located at the end ofTM6 and its side chain is expected to exhibit relatively high con-formational flexibility, whereas K2736.35 is located in the trans-membrane core and its side chain can interact with neighboringresidues I2776.39 and R3287.55 (Fig. 4 E and F). Upon agonistbinding, there is a downfield shift in the peak representing

44.544.5

45.045.0

45.545.5

46.046.0

K60+carazololK60+carazolol

44.544.5

45.045.0

45.545.5

46.046.0

K60 apoK60 apo

44.544.5

45.045.0

45.545.5

46.046.0

K60+BIK60+BI

44.544.5

45.045.0

45.545.5

46.046.0

K60+BI+GsK60+BI+Gs

30.030.0

2.752.752.852.852.952.95

10.010.0

20.020.0

0.00.0

TM1 K60TM1 K60

45.1447 ppm45.1447 ppm

44.544.5

45.045.0

45.545.5

46.046.0

K333+carazololK333+carazolol

44.544.5

45.045.0

45.545.5

46.046.0

K333 apoK333 apo

44.544.5

45.045.0

45.545.5

46.046.0

K333+BIK333+BI

44.544.5

45.045.0

45.545.5

46.046.0

K333+BI+GsK333+BI+Gs

2.652.65 2.702.702.802.802.902.903.003.000.00.0

10.010.0

20.020.0

30.030.0

40.040.0

H8 K333H8 K333

45.1612 ppm45.1612 ppm

Peak 1Peak 1

Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

K60K601.591.59R333KR333K8.518.51

E338E3388.568.56

L339L3398.578.57

F336F3368.548.54F332F3328.508.50

L64L6412.5012.50

F61F611.601.60

L53L531.521.52

K60K601.591.59R333KR333K8.518.51

E338E3388.568.56

L339L3398.578.57

F336F3368.548.54 F332F3328.508.50

L64L6412.5012.50

F61F611.601.60

L53L531.521.52

K60K601.591.59

R333KR333K8.518.51

H8H8

E338E3388.568.56

F61F611.601.60

L64L6412.5012.50

L339L3398.578.57

F332F3328.508.50

K60K601.591.59

R333KR333K8.518.51

H8H8

E338E3388.568.56

F61F611.601.60

L64L6412.5012.50

L339L3398.578.57

F332F3328.508.50

CBA

D

E

F

Fig. 2. Conformational changes of TM1 and H8 during β2AR activation. (A and B) The 1H-13C HSQC spectra of K601.59 from TM1 (A) and K3338.51 from H8 (B).The one-dimensional (1D) slices of corresponding 1H-13C HSQC spectra in H dimension are shown at the bottom. (C–E) The local environments of the twoprobes are shown by side views in inactive (gray, C) and active conformations (green, D), as well as cytoplasmic views in inactive (gray, E) or active con-formations (green, F). The agonist BI-167107 is abbreviated by BI.

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K2676.29. There are two peaks for K2676.29 in the β2AR-Gs com-plex (Fig. 4B). Peak 2 has the same chemical shift seen with ag-onist alone but with marked reduction in intensity. Of note, thedynamics of the cytoplasmic end of TM6 may impact the cyto-plasmic end of TM5, given their close proximity (Fig. 4F). K2275.56

in TM5 is close enough to have a polar interaction with E2686.28

(Fig. 4F). Thus, the minor Peak 1 in K2275.56 and in K2676.29 mayreflect different TM5–TM6 interactions in a small population ofnucleotide-free β2AR-Gs complex.It is interesting to note that the spectra of K2736.35 and

R328K7.55 are similar (Fig. 4 C and D). This may be due to theclose proximity of K2736.35 and R3287.55 observed in the inactivestate crystal structures (Figs. 1 C and D and 4E) where R328K7.55

could form a hydrogen bond with the backbone carbonyl ofK2706.32. This interaction would be lost upon coupling with Gs.For both K2736.35 and R328K7.55, agonist binding leads to adownfield shift in Peak 1, and Peak 1 is lost in the β2AR-Gs com-plex. Peak 2 may represent a conformation that is more exposed tothe solvent, therefore Peak 2 is less affected by agonist-binding or Gprotein coupling than Peak 1.

Conformational Differences in Gs and Gi1 Coupling. Recently, anumber of GPCR-Gi/o complex structures have been determinedby cryo-EM (5–7, 25–28). The Gs and β2AR interaction interfaceis composed by TM3, TM5, TM6, and ICL2. While Gαi/o inter-actions with TM3, TM5, and TM6 are similar in GPCR-Gi/oprotein complexes, TM2, TM7, and H8 are also involved information of the A1 adenosine receptor (A1R)-Gi complex, andICL2 and the junction between TM7 and H8 contribute to for-mation of the rhodopsin-Gi complex (SI Appendix, Fig. S5). ForGPCR-Gi/o complexes, the outward displacement of TM6 is

smaller than observed for the β2AR-Gs complex. The differencesin the interaction interface for these complexes might contributeto G protein coupling preferences. As noted above, the β2ARcan couple to both Gs and Gi. However, efforts toward obtainingthe structure of β2AR-Gi complex have failed due to the rela-tively weak interaction and instability of the complex. As a con-sequence, little is known about differences in the interactionsbetween β2AR and Gi compared with the β2AR-Gs complex.To provide more structural insights into the basis of β2AR

coupling selectivity for Gs over Gi, we next sought to compareβ2AR binding with Gs and Gi by using the 1H-13C HSQC spec-tra of the β2AR-BI-167017-Gi1

EMPTY complex (Fig. 5). Theβ2AR-Gi1

EMPTY complex was formed by adding the nucleotid-ase apyrase after complex formation to degrade any releasedGDP. Fig. 5 compares the spectra of β2AR-Gs

EMPTY andβ2AR-Gi1

EMPTY complexes. Consistent with the previous obser-vation that the β2AR-Gi1 complex is less stable than the β2AR-Gscomplex (29), we observed smaller spectral changes in the β2AR-Gi1 complex sample. The NMR spectral changes of K2275.56,K2676.29, K2736.35, and R328K7.55 corresponding to the conforma-tional changes of TM5, 6, and 7 are similar to those obtained fromthe β2AR-Gs complex; however, peak 2 of K2275.56 and K2736.35

and peak 1 of K2676.29 are notably weaker in the β2AR-Gi1 complexcompared to the β2AR-Gs complex. However, it’s hard to discrim-inate whether the TM6 outward displacement of β2AR−Gi1 issmaller than that of β2AR−Gs based on the signal intensitydifference observed by NMR.Remarkably, there are nearly no chemical shift changes for

K14034.52 in ICL2 and K1474.39 in TM4 in the β2AR-Gi1 complex,which is in contrast to what is observed for β2AR-Gs complex(Fig. 5 B and C). The observed decrease of peak intensity is likely

44.544.5

45.045.0

45.545.5

46.046.0

K140+carazololK140+carazolol44.544.5

45.045.0

45.545.5

46.046.0

K147+carazololK147+carazolol

44.544.5

45.045.0

45.545.5

46.046.0

K140 apoK140 apo44.544.5

45.045.0

45.545.5

46.046.0

K147 apoK147 apo

44.544.5

45.045.0

45.545.5

46.046.0

K140+BIK140+BI44.544.5

45.045.0

45.545.5

46.046.0

K147+BIK147+BI

44.544.5

45.045.0

45.545.5

46.046.0

K140+BI+GsK140+BI+Gs44.544.5

45.045.0

45.545.5

46.046.0

K147+BI+GsK147+BI+Gs

2.752.752.852.852.952.953.053.050.00.0

20.020.040.040.060.060.080.080.0

100.0100.0

2.702.702.802.802.902.903.003.000.00.0

10.010.0

20.020.0

30.030.0

TM4 K147TM4 K147ICL2 K140ICL2 K140

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

45.2171 ppm45.2171 ppm 45.1886 ppm45.1886 ppm

K147K1474.394.39

Q65Q6512.5112.51T66T662.372.37

Y70Y702.412.41

2.92.9Å

TM4TM2TM2TM1TM1

Q65Q6512.5112.51

T66T662.372.37 Y70Y702.412.41

K147K1474.394.39

TM4TM4TM2TM2TM1TM1

K140K14034.5234.52

ICL2ICL2

F139F13934.5134.51

TM5TM5TM4TM4 TM3TM3

K147K1474.394.39

Peak 1Peak 1

Q229Q2295.585.58

4.2 4.2 Å

K140K14034.5234.52

ICL2ICL2

K147K1474.394.39

Q229Q2295.585.58

F139F13934.5134.51

V217V217 H41H41F376F376

R380R380Q383Q383

TM5TM5TM4TM4 TM3TM3

A B C

D

E

F

Fig. 3. Conformational changes of ICL2 from inactive to active state. (A and B) 1H-13C HSQC spectra of K14034.52 (A) and K1474.39 (B). The 1D slices ofcorresponding 1H-13C HSQC spectra in H dimension are shown at the bottom. The local environment of the two probes are shown by side views in inactive(gray, C, E) and active (green, D, F) conformations. Gαs is shown in purple (D). The agonist BI-167107 is abbreviated by BI.

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44.544.5

45.045.0

45.545.5

46.046.0

K227+carazololK227+carazolol

44.544.5

45.045.0

45.545.5

46.046.0

K227 apoK227 apo

44.544.5

45.045.0

45.545.5

46.046.0

K227+BIK227+BI

44.544.5

45.045.0

45.545.5

46.046.0

K227+BI+GsK227+BI+Gs

2.702.702.802.802.902.903.003.000.00.05.05.0

10.010.015.015.020.020.025.025.0

TM5 K227TM5 K227

45.1184 ppm45.1184 ppm

44.544.5

45.045.0

45.545.5

46.046.0

K267+carazololK267+carazolol44.544.5

45.045.0

45.545.5

46.046.0

K273+carazololK273+carazolol

44.544.5

45.045.0

45.545.5

46.046.0

K267 apoK267 apo44.544.5

45.045.0

45.545.5

46.046.0

K273 apoK273 apo

44.544.5

45.045.0

45.545.5

46.046.0

K267+BIK267+BI44.544.5

45.045.0

45.545.5

46.046.0

K273+BIK273+BI

44.544.5

45.045.0

45.545.5

46.046.0

K267+BI+GsK267+BI+Gs44.544.5

45.045.0

45.545.5

46.046.0

K273+BI+GsK273+BI+Gs

20.020.0

40.040.0

60.060.0

2.652.652.752.752.852.852.952.950.00.02.02.04.04.06.06.08.08.0

10.010.0

2.502.502.602.602.702.702.802.802.902.903.003.00

TM6 K267TM6 K267 TM6 K273TM6 K273

45.0411 ppm45.0411 ppm 45.1809 ppm45.1809 ppm

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

F

44.544.5

45.045.0

45.545.5

46.046.0

K328+carazololK328+carazolol

44.544.5

45.045.0

45.545.5

46.046.0

K328 apoK328 apo

44.544.5

45.045.0

45.545.5

46.046.0

K328+BIK328+BI

44.544.5

45.045.0

45.545.5

46.046.0

K328+BI+GsK328+BI+Gs

20.020.0

2.652.652.752.752.852.852.952.950.00.0

5.05.0

10.010.0

15.015.0

TM7 K328TM7 K328

46.2632 ppm46.2632 ppm

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

K2275.71

TM5TM6

K2676.29

K2736.35

TM7

H8

R328K7.55

K2706.32

E2686.28

Gs a5Gs a5K227K2275.715.71

TM5TM5 TM6TM6

K267K2676.296.29

K273K2736.356.35

TM7TM7

H8H8

R328KR328K7.557.55

K270K2706.326.32

E268E2686.286.28

K22K22K22K22K22K22K22K22K22K22K22K22K2277777775.71

TMTTTTTMTMTTTTTTMTMTMTMM5TTTMTMMTMTMMTTMTMMTMMMMTMMMTM6

K2676.29

KKK27K27K27K27K2K2727K27272727K27K27K27K272K27K27777K27K27K27K27227K27K27K27K27K27K27K27KK27K27777K27K22222KK 333333333333333333333333333333333333333333366666666.36.33356666.36.36666.336.3336.3

TM77777

H8H8H8H8H8H8H8H8H8HH8888888H8H8H8H888H8H888888H8H88888H8888H88H8H88888

R32R32R32R32R32R32R32R32R32R32R323R3 8K7.55

K2706.326.66 333333336.3.3..3336 3.36.336 3..3333.3.3.3

E26666666666666688888888888888888888888888888888888886 2866.2666 286.28666 286.286.28.286 286.286 28886.288666 2866.2828886.286 286 888886 866 28886 886

K227K2275.715.71

TM5TM5TM6TM6

K267K2676.296.29

K273K2736.356.35

TM7TM7

H8H8

R328KR328K7.557.55

K270K2706.326.32

E268E2686.286.28

E

A B C D

Fig. 4. Conformational changes of TM5, TM6, and TM7 during β2AR activation. (A–D) The 1H-13C HSQC spectra of K2275.71 (A), K2676.29 (B), K2736.35 (C), andK3287.55 (D). The 1D slices of corresponding 1H-13C HSQC spectra in H dimension are shown at the bottom. The local environments of the four probes areshown in inactive (gray, E) and active (green, F) conformations. The α5 helix from Gsα is shown as purple cartoon. The agonist BI-167107 is abbreviated by BI.

Ma et al. PNAS | September 15, 2020 | vol. 117 | no. 37 | 23101

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TM1 K60TM1 K60

44.544.5

45.045.0

45.545.5

46.046.0

2.652.652.752.752.852.852.952.95

10.010.0

20.020.0

2.702.702.802.802.902.903.003.000.00.0

10.010.0

20.020.0

30.030.0

2.752.752.852.852.952.953.053.050.00.0

20.020.040.040.060.060.080.080.0

100.0100.0

2.702.702.802.802.902.903.003.000.00.05.05.0

10.010.015.015.020.020.025.025.0

45.1447 ppm45.1447 ppm 45.2171 ppm45.2171 ppm 45.1184 ppm45.1184 ppm45.1886 ppm45.1886 ppm

TM4 K147TM4 K147ICL2 K140ICL2 K140

44.544.5

45.045.0

45.545.5

46.046.0

44.544.5

45.045.0

45.545.5

46.046.0

TM5 K227TM5 K227

44.544.5

45.045.0

45.545.5

46.046.0

0.00.0

30.030.0

K60+BI+GsK60+BI+GsK60+BI+GiK60+BI+Gi

K140+BI+GsK140+BI+GsK140+BI+GiK140+BI+Gi

K147+BI+GsK147+BI+GsK147+BI+GiK147+BI+Gi

K227+BI+GsK227+BI+GsK227+BI+GiK227+BI+Gi

A B C D

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2Peak 1Peak 1

Peak 2Peak 2

44.544.5

45.045.0

45.545.5

46.046.0

K60+BIK60+BIK60+BI+GsK60+BI+Gs

44.544.5

45.045.0

45.545.5

46.046.0

K60+BIK60+BIK60+BI+GiK60+BI+Gi

44.544.5

45.045.0

45.545.5

46.046.0

K140+BIK140+BIK140+BI+GsK140+BI+Gs 44.544.5

45.045.0

45.545.5

46.046.0

K147+BIK147+BIK147+BI+GsK147+BI+Gs

Peak 1Peak 1Peak 2Peak 2

44.544.5

45.045.0

45.545.5

46.046.0

K140+BIK140+BIK140+BI+GiK140+BI+Gi 44.544.5

45.045.0

45.545.5

46.046.0

K147+BIK147+BIK147+BI+GiK147+BI+Gi

44.544.5

45.045.0

45.545.5

46.046.0

K227+BIK227+BIK227+BI+GsK227+BI+Gs

44.544.5

45.045.0

45.545.5

46.046.0

K227+BIK227+BIK227+BI+GiK227+BI+Gi

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1

Peak 1Peak 1Peak 2Peak 2

Peak 1Peak 1Peak 2Peak 2

2.502.502.602.602.702.702.802.802.902.903.003.000.00.0

20.020.0

40.040.0

60.060.0

2.652.652.752.752.852.852.952.95

2.02.04.04.06.06.08.08.0

10.010.0

2.652.652.752.752.852.852.952.950.00.0

5.05.0

10.010.0

15.015.0

2.702.702.802.802.902.903.003.000.00.0

10.010.0

20.020.0

30.030.0

40.040.045.0411 ppm45.0411 ppm 45.1809 ppm45.1809 ppm 45.2632 ppm45.2632 ppm 45.1612 ppm45.1612 ppm

TM6 K267TM6 K267 TM6 K273TM6 K273

44.544.5

45.045.0

45.545.5

46.046.0

44.544.5

45.045.0

45.545.5

46.046.0

TM7 K328TM7 K328 H8 K333H8 K333

44.544.5

45.045.0

45.545.5

46.046.0

44.544.5

45.045.0

45.545.5

46.046.0

K267+BI+GsK267+BI+GsK267+BI+GiK267+BI+Gi

K273+BI+GsK273+BI+GsK273+BI+GiK273+BI+Gi

K328+BI+GsK328+BI+GsK328+BI+GiK328+BI+Gi

K333+BI+GsK333+BI+GsK333+BI+GiK333+BI+Gi

E F G H

Peak 1Peak 1Peak 2Peak 2Peak 1Peak 1Peak 2Peak 2

44.544.5

45.045.0

45.545.5

46.046.0

K267+BIK267+BIK267+BI+GsK267+BI+Gs 44.544.5

45.045.0

45.545.5

46.046.0

K273+BIK273+BIK273+BI+GsK273+BI+Gs

Peak 1Peak 1Peak 2Peak 2 Peak 1Peak 1Peak 2Peak 2

44.544.5

45.045.0

45.545.5

46.046.0

K267+BIK267+BIK267+BI+GiK267+BI+Gi 44.544.5

45.045.0

45.545.5

46.046.0

K273+BIK273+BIK273+BI+GiK273+BI+Gi

44.544.5

45.045.0

45.545.5

46.046.0

K328+BIK328+BIK328+BI+GsK328+BI+Gs 44.544.5

45.045.0

45.545.5

46.046.0

K333+BIK333+BIK333+BI+GsK333+BI+Gs

44.544.5

45.045.0

45.545.5

46.046.0

K328+BIK328+BIK328+BI+GiK328+BI+Gi 44.544.5

45.045.0

45.545.5

46.046.0

K333+BIK333+BIK333+BI+GiK333+BI+Gi

Peak 1Peak 1Peak 2Peak 2Peak 1Peak 1

Peak 2Peak 2

Fig. 5. Comparison of conformational changes in β2AR coupled to Gs or Gi1. BI is short for BI-167107. (A–H) The 1H-13C HSQC spectra of the eight probes fromTM1 to H8. The eight probes are K601.59 (A), K14034.52 (B), K1474.39 (C), K2275.56 (D), K2676.29 (E), K2736.35 (F), R328K7.55 (G), and R333K8.51 (H). For each probe,the overlay of the 1H-13C HSQC spectra of agonist-bound β2AR (green) and β2AR-GsEMPTY (blue) are shown at the top, followed by the comparison of the 1H-13C HSQC spectra of agonist-bound β2AR (green) and β2AR-Gi1EMPTY (pink), then by the overlay of β2AR-GsEMPTY (blue) and β2AR-Gi1EMPTY (pink). The 1D slicesof corresponding 1H-13C HSQC spectra in H dimension are shown at the bottom.

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due to the increase in mass. The lack of spectral changes forK14034.52 and K1474.39 suggest that ICL2 does not form an alphahelix when coupled to Gi1 and may have only weak interactionswith Gi1, possibly explaining the less efficient coupling and theoverall instability of the β2AR-Gi1 complex.As noted above, the insertion of F13934.51 in ICL2 of the β2AR

into a hydrophobic pocket formed by H41, V217, F376, C379,and R380 in the Gαs is essential for GDP release (SI Appendix,Figs. S6A and S7A). Of these, only F376 is conserved as F336 inGi (SI Appendix, Fig. S6 B and C). When comparing sequences ofICL2 from GPCRs that couple to Gs, Gi/o, Gq/11, and G12, po-sition 34.51 is most often a L, F, I, or M for Gs coupled receptors,while a broader range of amino acids are found in Gi/o coupled

receptors (SI Appendix, Fig. S6D). It is possible that interactionswith 34.51 in ICL2 may be less important for coupling with Gi1than Gs. This is in agreement with the recent structures of Gi incomplex with the cannabinoid receptor subtype 1 (CB1) (27), theμ-opioid receptor (μOR) (6), and the A1R (7). For these recep-tors, the interactions between the amino acid at position 34.51 inICL2 (L in CB1 and A1R, and V in μOR) and Gi are muchweaker, interacting with only one or two side chains of Gi (SIAppendix, Fig. S7 B–D). However, this weaker interaction betweenICL2 and Gi is not universal. For the neurotensin receptor subtype1 (NTSR1), which couples promiscuously to Gi/o, Gs, and Gq/11(30), F17434.51 in ICL2 packs into a pocket formed by four aminoacids in Gi (SI Appendix, Fig. S7E). The importance of the amino

A

C D

B

E F

AR-GiAR-Gi

AR-GiAR-Gi

AR-GiAR-Gi

G ( 5)

Fig. 6. Comparison of conformational changes measured by MD simulations in β2AR coupled to Gs or Gi. Backbone rmsd in Å extracted from 3 × 3-μs-long MDsimulations of β2AR-Gi (pink) and β2AR-Gs (cyan) of structural elements at the receptor-G protein interface. (A) ICL1 of β2AR: E6212.48-Q6512.51. (B) ICL2 of β2AR:I1353.54-A1504.42. (C) H8 of β2AR: S3298.47-C3418.59. (D) α5 helix of Gαi and Gαs. (E) TM5 of β2AR: N1965.35-R239ICL3. (F) TM6 of β2AR: C2656.27-Q2996.61. The rmsdvalues were calculated relative to the respective structural element in the starting structure of β2AR-GiEMPTY and β2AR-GsEMPTY.

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acid in position 34.51 was initially demonstrated for the Gq/11coupled M1 muscarinic receptor (M1R) where the substitution ofL13134.51 for Ala in ICL2 led to a loss of Gq/11 coupling (24). Inthe recent cryo-EM structure of the M1R-G11 complex, L13134.51

packs into a pocket formed by four amino acids in G11 (28) (SIAppendix, Fig. S7F). Like Gs coupled GPCRs, L, F, I, or M aremore commonly found at position 34.51 in Gq/11 coupled GPCRs(SI Appendix, Fig. S6D). While the evidence suggests that weakinteractions between F13934.51 in ICL2 of the β2AR and Gi1 mayaccount for poor coupling efficiency, we cannot exclude the pos-sibility that β2AR simply couples less efficiently to Gi and the lackof strong engagement with ICL2 is a manifestation of that.Similar to Gs, Gi1 binding also has little effect on R333K8.51

located at H8 (Fig. 5H). The decrease in intensity is likely due tothe increased size of the complex. Notably, the decrease in in-tensity is greater for Gs

EMPTY compared to Gi1EMPTY, consistent

with the formation of a more stable complex. The low intensitydownfield peak observed in R333K8.51 with Gs is not observedwith Gi1. The spectra of K601.59 in TM1 are similar for Gs andGi1, with a reduction of intensity and the peak splitting into aslightly upfield-shifted peak and a smaller downfield peak(Fig. 5A). ICL1 and H8 are not observed to interact with Gs inthe β2AR-Gs complex; however, the orientation of ICL1 relativeto H8 changes upon complex formation with the loss of anelectrostatic interaction between K601.59 in ICL1 and R328K7.55

in H8.We have previously observed evidence for a transient inter-

mediate state following the disruption of the β2AR-Gs complexby the addition of GDP (8). In an effort to detect this state byNMR spectroscopy, we formed β2AR-Gs and β2AR-Gi1 com-plexes in the presence of 300 μM GDP. As can be seen in SIAppendix, Figs. S8 and S9, we don’t observe a distinct state in thepresence of GDP. The spectra are consistent with a mixture ofnucleotide-free β2AR-Gs or β2AR-Gi1, and β2AR bound to ag-onist alone. Our inability to detect a distinct GDP-bound β2AR-Gs complex could be due to the insensitivity of our probes or tothe fact that the amount of the intermediate may be too smallto detect.

Molecular Dynamics Simulations Showed Different ICL2 Behavior inthe β2AR-GiEMPTY and the β2AR-GsEMPTY Complex. To investigate thedynamics at the receptor G protein interface, classical unbiasedMD simulations of the β2AR-Gi1

EMPTY and the β2AR-GsEMPTY

complex were carried out. The β2AR-Gi1EMPTY model was built

based on the β2AR-GsEMPTY complex (Protein Data Bank [PDB]

ID code: 3SN6) (see Methods for more details) and as a result,ICL2 in the starting structures of both β2AR-Gi1

EMPTY andβ2AR-Gs

EMPTY complex are in an alpha-helical conformation.Three independent, 3-μs-long molecular dynamics (MD) simu-lations were carried. While the simulations are too short to ob-serve unfolding of the secondary structure, the significantlyhigher rmsd of the ICL2 region of the β2AR-Gi1 complex com-pared to the β2AR-Gs complex suggests the starting alpha-helicalstructure of ICL2 is less stable in the β2AR-Gi1

EMPTY structure.Of note, such changes are not observed for ICL1, H8, nor Gα(α5)when both systems are compared (Fig. 6 A–D). The only otherstructural elements at the receptor G protein interface with in-creased rmsd values are TM5 and TM6 (Fig. 6 E and F),

consistent with the high dynamics of their cytoplasmic ends ob-served by chemical shift analysis in β2AR-Gi1

EMPTY and in β2AR-Gs

EMPTY complexes. A close inspection of the MD simulationsresults suggests a higher flexibility of K14034.52 and K1474.39 fromICL2 for the β2AR-Gi1

EMPTY compared to ICL2 from β2AR-Gs

EMPTY as measured by the average rms fluctuation (RMSF)(SI Appendix, Fig. S10). The average RMSF value for K14034.52 istwo times higher and for K1474.39 is six times higher in β2AR-Gi1

EMPTY than β2AR-GsEMPTY. The MD simulations results are

in agreement with the NMR experimental observation that ICL2plays different roles when β2AR couples to Gi or Gs.

DiscussionWe applied NMR spectroscopy to monitor structural changes inthe cytoplasmic surface of the unliganded β2AR and in responseto an inverse agonist, an agonist, Gs, and Gi. We have beenunable to obtain crystal or cryo-EM structures of the apo-β2ARand agonist-bound β2AR due to its instability and possibly due toconformational heterogeneity. Our NMR studies provide furtherevidence for conformational heterogeneity as we observe morethan one peak for K1474.39, K2736.35, and K3287.55 in apo andagonist-bound β2AR (Figs. 2–4). Agonist binding leads tochanges in K1474.39 in ICL2, K2676.29, and K2736.35 in TM6, andin K3287.55 in TM7; however, additional changes are observedupon the addition of Gs and Gi1. This suggests that for the β2ARagonist, binding alone cannot fully stabilize the G protein boundconformation, as has been observed in previous fluorescence anddouble electron electron resonance spectroscopy experiments(8, 13). The structural changes stabilized by Gi1 and Gs arequalitatively the same for TM5, TM6, and TM7; however, ourNMR studies and supporting MD simulations suggest that Gi1does not promote the formation of an alpha helix in ICL2.

MethodsThe receptors were expressed in Sf9 insect cells, purified by affinity columnand size exclusion chromatography, and labeled by reductive methylation.NMR data were collected at 25 °C on a Burker Avance 800-MHz spectrometerequipped with a cryoprobe. The 1H-13C HSQC spectra were recorded withspectral width of 11,160.71428 Hz in the 1H-dimension (w1) and 14,492.7536 Hzin the 13C-dimension (w2) centered at 46 ppm. For all spectra, 1024 × 256complex points were recorded and a relaxation delay of 2 s was used to allowspin to relax back to equilibrium. MD simulations were performed usingGromacs simulation package. Further details are proved in SI Appendix.

Data Availability. All study data are included in the article and SI Appendix.

ACKNOWLEDGMENTS. This work was supported by the Beijing AdvancedInnovation Center for Structural Biology, Tsinghua-Peking Joint Center forLife Science, Tsinghua University. C.J. acknowledges funding from theNational Key R&D Program of China (Grant 2016YFA0501201). P.W.H.acknowledges funding by the Deutsche Forschungsgemeinschaft (GermanResearch Foundation) through SFB1423, project 421152132 and HI1502/1-2,project 168703014; the Stiftung Charitè; and the Einstein Center Digital Fu-ture. All NMR spectra were obtained at the Beijing NMR Center and the NMRfacility of the National Center for Protein Sciences at Peking University. Wethank Jiawei Zhao for providing G protein P1 virus and suggestions. Wethank the Gauss Center for Supercomputing (GCS) e.V. for funding this proj-ect by providing computing time on the GCS Supercomputer SuperMUC atLeibniz Supercomputing Centre. B.K.K. is a Chan Zuckerberg BiohubInvestigator.

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