G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β 2 -adrenergic receptor (β 2 AR) using 19 F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β 2 AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Proteins display a range of motions associated with function, from pico- to nanosecond timescale amino acid side-chain reorientations to inter-domain motions that may happen on the millisecond to second timescale (). Although such protein dynamics are likely important for the signaling versatility and allosteric regulation of GPCRs, the dynamic properties of GPCRs remain poorly understood. Crystallography typically captures the lowest energy states within an ensemble of conformations. Other methods are therefore required to characterize transiently populated conformations as well as the transitions between different conformations. Using NMR spectroscopy ofCH-ε-methionines, we recently observed significant conformational heterogeneity in the transmembrane core of βAR while bound to agonist and inverse agonist, as well as evidence of conformations not observed in crystal structures (). Here, we extend these studies by assessing βAR conformational dynamics in the cytoplasmic, G-protein-coupling domain of the receptor. We useF NMR spectroscopy of fluorine-labeled βAR to identify representative states and exchange rates between these states as a function of ligand efficacy. To provide a structural framework for this conformational heterogeneity, we utilize pulsed electron paramagnetic resonance spectroscopy (double electron-electron resonance, or DEER) of nitroxide spin-labeled βAR.

G-protein-coupled receptor signaling relies on allosteric coupling between the extracellular facing ligand-binding pocket and the cytoplasmic domain of the receptor. Ligands may activate a signaling pathway (agonists), inhibit the basal level of signaling (inverse agonists), or bind but not perturb signaling (neutral antagonists), all by changing the conformational ensemble of a GPCR. Recent X-ray crystal structures of the βAR have provided high-resolution insight into two conformations associated with GPCR function: an inactive, inverse agonist-bound state and the active state in complex with an agonist and the G protein G). These structures reveal how subtle changes in the ligand-binding pocket translate into a 14 Å outward displacement of transmembrane 6 (TM6) in the cytoplasmic domain of the receptor ( Figure 1 A) ().

(C) For DEER spectroscopy, β 2 AR was labeled at the cytoplasmic ends of TM4 (site N148C-IAP) and TM6 (site L266C-IAP) with the nitroxide label 3-(2-iodoacetamido)-2,2,5,5-tetramethylpyrroline-1-oxyl (IA-PROXYL).

(B)F-NMR studies utilize the fluorine label 2-bromo-4-(trifluoromethyl)acetanilide (F-BTFA) that reports changes in the chemical environment at the cytoplasmic end of TM6. See Figure S1 and Table S1 for construct design and validation.

(A) Comparison of crystal structures of inactive, carazolol-bound, and active β 2 AR in complex with agonist BI167107 and G s . The crystal structures reveal a 14 Å outward displacement of TM6 upon β 2 AR activation. Cys265, used for 19 F-NMR experiments, is highlighted in spheres.

To determine whether the failure of isoproterenol to fully stabilize the active state is due to dissociation/association kinetics, we examined the response to the ultra-high-affinity agonist BI167107. In bothF-NMR and DEER experiments, we observe substantially more of the S3 conformation of βAR ( Figures 5 E and 5F). However, even bound to this high-affinity agonist, ∼40%–60% of the receptor is in conformations comprised of the inactive S1 and S2 states. As noted above, due to the very slow dissociation kinetics, it is unlikely that this observed conformational heterogeneity results from βAR molecules not bound to BI167107. The reduced signal representing S1 and S2 does not allow accurate measurement of relaxation dispersion by CPMG experiments. However, we observed slow exchange between active (S3) and inactive (S1 and S2) conformations inF-NMR saturation transfer experiments. Saturation of theF-NMR peak originating from the inactive state S1 led to a decrease in signal of the peak originating from the active intermediate S3, suggesting that inactive and active conformations exchange on a slow timescale ( Figures 5 G and 5H). Through control experiments, which allowed us to identify the extent of off-resonant saturation ( Figures 5 H and S5 C), the exchange resulting from saturating the inactive ensemble is consistent with the lifetime of the S3 state to be 660 ms. The kinetics of these transitions is faster than our previous fluorescence studies examining the activation of purified βAR by isoproterenol (). In those experiments, the change in fluorescence associated with receptor activation occurs in two phases, with tvalues of 2.5 s and 150 s. While these experiments highlighted the slow transition associated with receptor activation, the studies were done under non-steady-state conditions in which ligand binding and unbinding as well as receptor conformational changes contribute to the change in fluorescence. In the NMR kinetics experiment presented in Figure 5 H, the receptor is at equilibrium between inactive and active states, and the agonist BI167107 has such exceptionally slow binding/unbinding kinetics that they do not contribute to the observed rate of transitions between inactive and active states. As such, the saturation transfer experiment here directly shows a high-energy barrier between the inactive and S3 states that may be responsible for the slower transition to the active state observed for the βAR and other GPCRs as compared to rhodopsin ().

Based on the area under the S3 peak for isoproterenol bound βAR, this state represents approximately 15% of the total receptor population and is consistent with the fraction of receptor in the active-like state observed in DEER studies. Additionally, we performed lineshape simulations of the major peak arising from S1 and S2 states using estimates of receptor exchange kinetics derived from CPMG experiments ( Figures 5 C, 5D, and S5 A). Such analysis shows that isoproterenol increases the fraction of βAR in the S2 state with a broken ionic lock, which is consistent with the proportions observed in DEER studies. Notably, due to the difference in population observed for S1 and S2 for isoproterenol-bound receptor, the calculated lifetimes differ for each state ( Figure 3 C). The S1 lifetime is 394 ± 55 μs, which is similar to that observed for carazolol-bound receptor. On the other hand, the lifetime of the S2 state is 756 ± 105 μs, similar to that for unliganded receptor. These observed differences in lifetimes of the S1 and S2 states between agonist, inverse agonist, and unliganded receptor are shown in Figure 3 C.

(C) Saturation transferF-NMR spectra for βAR bound to BI167107 with varying lengths of saturating continuous wave irradiation. To saturate signals arising from the S1+S2 state, CW irradiation was applied at −60.77 ppm, which is 420 Hz downfield of the S3 state at −61.47 ppm. To account for direct saturation of the S3 peak, identical CW irradiation was applied at −62.17 ppm, which is 420 Hz upfield of the S3 peak. The intensity of the S3 peak as a function of CW irradiation time is plotted in Figure 5 H.

We first assessed the ability of the lower-affinity full agonist isoproterenol to induce conformational changes in the βAR. Surprisingly, DEER experiments revealed that, even in the presence of a saturating concentration (2.5× molar excess, 0.5 mM) of a full agonist, most of the receptor remained in an inactive conformation, with ∼20% in a conformation similar to the fully active βAR bound to BI167107 and Nb80 ( Figure 5 A). Isoproterenol also appears to increase the fraction of receptor populating a conformation consistent with a broken ionic lock when compared with unliganded and inverse agonist-bound receptor. To determine whether the inactive state peaks represent non-functional receptor, we added Nb80 and observed that most of the protein transitions to an active state ( Figure 5 A). NMR studies revealed a more complex set of conformations associated with agonists, perhaps due to the sensitivity of theF-NMR probe to local conformation. We observe a new upfield peak in theF-NMR spectrum ( Figure 5 B) in the presence of saturating concentrations of isoproterenol. This new peak, labeled S3, has a chemical shift of −61.47 ppm, which is similar, but not identical, to the S4 state (−61.59 ppm) observed for βAR bound to BI167107 and Nb80 ( Figure 3 B). Addition of Nb80 to isoproterenol-bound βAR results in a predominant peak between S3 and S4 at −61.51 ppm ( Figure 5 B). Although Nb80 was added in 2.5× molar excess, the fast dissociation/association kinetics of isoproterenol may hinder complete stabilization of the S4 state. The resulting conformational heterogeneity is consistent with the increased peak linewidth observed for βAR bound to isoproterenol and Nb80 as compared to BI167107 and Nb80 (144 Hz and 114 Hz, respectively) as well as the small fraction of inactive receptor observed in the DEER distance distribution. Given the sensitivity of theF-NMR probe, we posit that the S3 peak represents an on-pathway intermediate toward the fully activated S4 conformation, which is adopted upon complete stabilization of the active state. In the experimental conditions presented here, this occurs only upon binding of a slowly dissociating agonist and Nb80. The fact that we cannot distinguish S3 from S4 by DEER spectroscopy may be due to limitations in sensitivity of this method at distances in the range of 50 Å. It is also possible that different conformations result in similar TM6-TM4 distances.

(G) Deconvolution of βAR+BI167107 spectrum into S1+S2 and S3 peaks. Arrows indicate positions of the spectrum irradiated in saturation transfer experiments with the resulting decay in signal shown in (H). See Figure S5 for saturation transfer NMR spectra.

(D) Simulation of S1 and S2 states for β 2 AR bound to isoproterenol and comparison with unliganded β 2 AR show an increase in the S2 state for isoproterenol-bound receptor.

(C) Deconvolution of β 2 AR+isoproterenol without Nb80 to highlight the S3 state. CPMG dispersion of the S1+S2 peak is shown in the inset.

(B) NMR spectrum of isoproterenol-bound βAR shows the presence of a new upfield peak corresponding to S3 (−61.47 ppm) as well as a peak originating from fast exchange of S1 and S2 states. Addition of Nb80 causes a transition to a peak between the S3 and S4 states (−61.51 ppm). The S4 state has a chemical shift of −61.59 ppm. See Figure S5 for isoproterenol lineshape analysis.

(A) Distance distributions for β 2 AR in the presence of isoproterenol alone and with Nb80. The dashed black trace represents the distribution from unliganded β 2 AR. DEER experiments do not provide sufficient resolution to distinguish S3 from S4.

To determine the effect of agonists on βAR structure and dynamics, we examined two full agonists: isoproterenol (760 nM), a catecholamine related to adrenaline, and BI167107 (84 pM). In the case of isoproterenol, association and dissociation kinetics are rapid (seconds to minutes) and may contribute to receptor dynamics; however, the dissociation kinetics of BI167107 are very slow (t= 30 hr) (), and this agonist will remain bound for the duration of the spectroscopic studies.

Although it is well established that agonists increase GPCR signaling by inducing a change in receptor conformation and ultimately leading to G-protein coupling, the mechanism associated with this allosteric process remains poorly understood. Crystal structures of βAR in a fully active conformation have relied on the presence of a protein bound to the intracellular surface to stabilize the active state. As a result, the degree of conformational changes induced by agonists alone remains poorly defined. In the absence of a stabilizing interaction with Nb80 or G, the βAR bound to a covalent agonist crystallized in an inactive conformation (). Additionally, molecular dynamics simulations of active, agonist-bound βAR in the absence of Nb80 or Gdemonstrate a rapid transition of the receptor to the inactive state (). These results, together with previous NMR studies (), suggest that the active conformation is not the lowest energy state for agonist-bound receptor. Here, we explore the effect of agonists on the structure of the cytoplasmic domain of the βAR in the absence of constraints imposed by a crystal lattice.

Most GPCRs, including the βAR, exhibit some degree of basal activity, suggesting that they are able to activate G proteins in the absence of agonist. The structural basis for basal activity is not known but may be due to the dynamic and flexible nature of the βAR such that a small fraction of receptor existing in an active state is capable of coupling to G. Inverse agonists like carazolol and ICI-118,551 would be expected to destabilize active states, either by reducing their equilibrium population or by decreasing the lifetime of states on the pathway to activation. Surprisingly, there is little difference in the steady-state DEER and NMR data between unliganded receptor and βAR bound to carazolol or ICI-118,551 ( Figures 4 A, 4B, S4 A, and S4B). The relative populations of S1 and S2 as determined by lineshape simulations are similar for unliganded and inverse agonist-bound receptor ( Figure 4 C). Notably, we do not reliably observe a peak corresponding to the active state in either DEER or NMR experiments of unliganded βAR, likely because such a transiently populated conformation is outside the current detection limit of these experiments. However, CPMG experiments show that the rate of interconversion between the S1 and S2 states is reduced by approximately half for unliganded receptor as compared with either carazolol-bound or ICI-118,551-bound receptor ( Figures 4 B, inset, and S4 B). The more rapid exchange between S1 and S2 in carazolol and ICI-118,551-bound receptor is illustrated in the energy landscape as a lower energy barrier between these two states ( Figure 1 D). This lower interconversion rate for unliganded receptor also results in an increased lifetime of both the S1 and S2 states, with a calculated value of 700 ± 137 μs ( Figure 3 C).

Using DEER spectroscopy, we confirmed that the change in theF-NMR spectrum occurs due to receptor activation ( Figure 2 D). The distance distribution shows a single conformation centered around 50 Å, consistent with the outward displacement of TM6 observed in the crystal structure of the βAR-BI167107-Nb80 complex. Simulation of the inter-nitroxide distance distribution for spin labels modeled in the βAR-BI167107-Nb80 crystal structure (PDB ID: 3P0G ) yielded good agreement with the observed DEER distance distribution ( Figures 2 D and 2E). Binding of a high-affinity agonist and an intracellular G-protein mimetic, therefore, fully stabilizes βAR in the conformation observed by X-ray crystallography. The absence of exchange dynamics inF-NMR CPMG experiments ( Figure 3 B, inset) suggests that this activated conformation in the presence of BI167107 and Nb80 is a relatively stable, low-energy conformation. In Figure 1 D, we depict the corresponding free-energy landscape, resulting from binding of agonist and Nb80, as being dominated by a single energy well, with a large energy barrier toward alternative conformations of the βAR. We refer to the fully active conformation with both agonist and Nb80 bound as S4. As the structure of Nb80-bound βAR is highly similar to that of the βAR-Gcomplex, the S4 state likely also represents the G-protein-coupled state of the receptor.

Activation of the βAR results in TM6 movement that creates a receptor-binding site for intracellular effector and regulatory proteins. This change was observed in crystal structures of βAR bound to BI167107 and either Gor Nb80 (). We utilized Nb80 as a G-protein surrogate in NMR and DEER experiments for two reasons ( Figure 2 D and Figure 3 B). First, generating homogeneously prepared βAR bound to Nb80 is significantly simpler as compared to G, as Nb80 binding does not depend on biochemically labile nucleotides. Second, Nb80 is significantly smaller than G(14 kDa versus 85 kDa), and this smaller size allowed us to generateF-NMR spectra of fully activated receptor with greater resolution due to longer transverse relaxation times. TheF-NMR spectrum of the βAR-BI167107-Nb80 complex shows a large change from the unliganded state, with the appearance of a new peak ( Figure 3 B). The upfield chemical shift of −61.59 ppm observed for βAR bound to Nb80 is consistent with greater solvent exposure of the cytoplasmic side of TM6 upon receptor activation. Additionally, the decreased peak linewidth is consistent with a single receptor conformation while bound to Nb80. Additionally, CPMG experiments revealed no evidence of millisecond timescale exchange dynamics ( Figure 3 B, inset).

In agreement with the structural heterogeneity observed in DEER experiments,F-NMR spectra show a broad line shape. Due to the slow dissociation rate of carazolol (tof dissociation = 30.4 hr) () compared to the timescale of the NMR experiment, ligand association and dissociation kinetics do not contribute to the observed structural heterogeneity and dynamics. In accord with molecular dynamics simulations, we observed the presence of high microsecond receptor exchange dynamics (exchange rate [k] = 6,200 ± 830 s) for carazolol-bound βAR in CPMG relaxation dispersion experiments ( Figure 3 A, inset). As a result of the exchange rate between the two ionic lock states, their unique chemical shifts are not observed in the spectra but are represented as a weighted average centered at −60.85 ppm due to classic exchange broadening and coalescence. However, knowing the exchange rate and assuming two states as suggested by the DEER distance distributions, it is possible to simulate the NMR lineshapes and chemical shifts for each of these states as they would appear in the absence of exchange (see Supplemental Experimental Methods ). The resulting simulated exchange-free spectra identify two states that likely correspond to the ionic lock states S1 and S2 ( Figures 3 A and S3 B). We assign the peak at −60.50 ppm to S1 (ionic lock intact) since more buried fluorine reporters are typically observed to be more downfield. Moreover, the linewidth of S2 (−61.30 ppm) in the absence of exchange is predicted to be broader based on the global lineshape simulations, hinting at greater heterogeneity in the ionic lock disrupted inactive state. For carazolol-bound βAR, both states are populated nearly equally, which is consistent with the results observed by DEER spectroscopy for the S1 and S2 states. Using these equilibrium populations for S1 and S2 with the exchange kinetics from the CPMG experiments, the calculated lifetime of the S1 and S2 states is 325 ± 44 μs ( Figure 3 C). Together, these results show that the inverse agonist carazolol does not fully stabilize TM6 in a single inactive conformation. In Figure 1 D, we represent the two states observed for carazolol-bound βAR as two energy wells of similar depth separated by a shallow energy barrier that allows the fast exchange between the two inactive conformations.

(B) The 19 F-NMR spectrum for β 2 AR bound to carazolol is shown in red. During NMR spectrum lineshape simulations, the chemical shift and inherent linewidths of S1 and S2 are allowed to vary but are constrained between all β 2 AR samples measured. Furthermore, the exchange rate is constrained to be that measured by CPMG experiments. The relative populations of S1 and S2 are determined for each sample. Here, we show the simulated spectrum for β 2 AR bound to carazolol (gray line) along with simulated spectra of S1 and S2 with varying rates of exchange. Due to the rapid exchange rate, the observed 19 F-NMR spectrum displays coalescence of the S1 and S2 states.

(A) Original NMR spectra for β 2 AR-Δ4 preparations are shown in a dashed line. For clarity and ease of visualization, the non-changing peak at −61.83 ppm (purple solid line) has been subtracted from each spectrum, yielding the colored spectra shown as a solid line in the main text.

(C) Lifetimes of the S1 and S2 states for β 2 AR were calculated using the measured exchange rates and the populations estimated by lineshape simulation. As the k ex could not be experimentally determined for β 2 AR bound to BI167107, there is potential for error in the simulated populations and lifetimes. The data are therefore illustrated in dotted lines. Error bars represent errors propagated from CPMG fits and determination of S1 and S2 populations. Generally, inverse agonists decrease the lifetime of both the S1 and S2 states, while agonists decrease the lifetime of the S1 state while preserving the lifetime of the S2 state.

(B) 19 F-NMR spectrum of β 2 AR bound to BI167107 and Nb80. The dashed green line serves as a marker for the S4 state. Inset shows the absence of CPMG relaxation dispersion for β 2 AR bound to BI167107 and Nb80.

(A)F-NMR spectrum of carazolol-bound βAR. The inset shows the presence of fast timescale dynamics, as assessed by CPMG relaxation dispersion profiles at two magnetic fields (500 MHz and 600 MHz). The simulated S1 and S2 peaks in the absence of exchange are shown in dotted lines, and the simulated combined lineshape arising from exchange of S1 and S2 is shown in gray. The simulated lineshapes are further illustrated in Figure S3 . The estimated errors in Rfor CPMG studies are smaller than the graphics used for illustration. Standard errors in kare dependent on errors in R, which were estimated by the spectral noise and variation in spectra between experiments from identically prepared samples. See Supplemental Experimental Methods for more details.

To provide a structural reference for NMR and DEER studies, we will first present results of experiments done under conditions used to obtain inactive and active state crystal structures. Carazolol is an ultra-high-affinity partial inverse agonist that reduces basal Gcoupling activity (). DEER spectroscopy revealed a broad distance distribution between TM6 and TM4 ( Figure 2 A). Modeling of the nitroxide spin labels in the carazolol-bound X-ray crystal structure of βAR (PDB ID: 4GBR ) () and simulation of the expected distance distribution (see Experimental Procedures ) showed substantial overlap with one of the populations observed in the DEER-derived distance distribution ( Figures 2 A and 2B). While the conformation of TM6 is similar in all inactive state crystal structures of the βAR, structural heterogeneity of TM6 in the inactive state has previously been observed in crystal structures of the βAR (), where one of two TM6 conformations resulted from different states of a highly conserved interaction between the DRY motif within TM3 and E285, termed the ionic lock. Although crystal structures of antagonist-bound βAR consistently show a broken ionic lock, molecular dynamics simulations indicate that the receptor transitions frequently between the two conformations (). In order to assess the theoretical DEER distance distribution for βAR in the ionic lock, we modeled the nitroxide labels onto the X-ray crystal structure of βAR with an intact ionic lock (PDB: 2YCW , chain A) at the same positions used for DEER studies of the βAR ( Figures 2 A and 2C) and simulated the inter-nitroxide distance distribution. This simulated distribution for βAR with an intact ionic lock overlaps one of the conformations experimentally observed for βAR. This ensemble with shorter distances may thus represent a population of the βAR with an intact ionic lock. We describe the inactive conformation with the ionic lock intact as S1 and the conformation with the ionic lock broken as S2. We observe a smaller population having shorter distances between nitroxide probes (28–33Å). These may reflect additional, less-populated conformational states and/or different rotamers of probes on the S1 conformation.

(D) Distance distribution for βAR bound to BI167107 and Nb80. The dashed green line serves as a marker for the S4 state. The gray line represents the simulated distance distribution for βAR bound to BI167107 and Nb80 using the previously determined crystal structure (PDB ID: 3P0G ), as shown in (E).

(C) Similar analysis as in (B) was performed on the structure of β 1 AR bound to carazolol but with an intact ionic lock. The red outline indicates rotamers modeled in (B). The mean distance for a state with an intact ionic lock is predicted to be shorter than for β 2 AR with a broken ionic lock.

(B) IA-PROXYL rotamers modeled onto βAR bound to carazolol (PDB ID: 4GBR ) using MTSSLWizard. The distance between these possible rotamers is then determined in a pairwise manner to yield the predicted distance distribution shown in A.

(A) Distance distribution for carazolol-bound βAR. The dotted lines show simulated nitroxide spin label distance distributions for a state with a broken ionic lock using carazolol-bound βAR (PDB ID: 4GBR ) and a state with an ionic lock intact using βAR (PDB ID: 2YCW ), as shown in (B) and (C).

We acquired spectra for βAR bound to saturating concentrations of four ligands: (1) carazolol, an ultra-high-affinity (32 pM) partial inverse agonist (); (2) ICI-118,551, a high-affinity (550 pM) full inverse agonist (); (3) isoproterenol, a low-affinity (229 nM) full agonist that is an analog of the endogenous hormone adrenaline; and (4) BI167107, an ultra-high-affinity full agonist (84 pM) (). Additionally, we acquired spectra for βAR bound to either agonist in the presence of the G protein mimetic Nb80. In total, NMR and DEER spectroscopy provide evidence for at least four distinct βAR states, which we label as S1, S2, S3, and S4 for the purpose of discussion ( Figure 1 D, top). While it is possible that the states identified by NMR and DEER spectroscopy are distinct and non-overlapping, the high level of agreement observed for equilibrium populations and their response to ligands strongly suggest that both techniques resolve similar conformations of the βAR. After assigning these states to specific structural conformations of the βAR, we illustrate insights into receptor function with energy landscape diagrams that change in the presence of inverse agonists, agonists, and Nb80 ( Figure 1 D). Although the energy diagram illustrations likely oversimplify the complexity of βAR dynamics, they show the key role that structural dynamics plays in GPCR function. Furthermore, the results suggest a marked difference in the conformational landscape of βAR as compared to the light-sensing GPCR rhodopsin. These differences in energetics may explain dissimilarities in signaling behavior between rhodopsin, which evolved for rapid and highly efficient detection of a photon, and hormone-activated GPCRs like the βAR, which evolved to have more complex signaling and regulatory behavior.

To provide a structural framework for the conformational heterogeneity observed inF-NMR spectra, we utilized DEER spectroscopy, which can measure distance distributions between two domains on a protein. For DEER spectroscopy, βAR was site-specifically labeled with nitroxide probes at L266C in TM6 and N148C in TM4 ( Figures 1 C, S1 A, S1D, and S1E), and the DEER data were analyzed to provide inter-nitroxide distance distributions using established methods ( Figure S2 ). Finally, we performed two additionalF-NMR relaxation experiments designed to examine the kinetics of conformational changes: CPMG relaxation dispersion experiments for fast microsecond to millisecond transitions and “hole burning” saturation-transfer experiments for slow transitions occurring on the 10–500 ms timescale (). Studies were performed with protein purified by ligand affinity chromatography to ensure that all spectroscopic data reflect functional states of the βAR ().

Background-subtracted dipolar evolution data are shown for each sample. The raw data is displayed as dots with the best fit shown in a solid line. Optimal fits of the DEER dipolar evolution strike a balance between smoothness, which suppresses peaks originating from artifacts, and resolution of individual distance peaks, which gives insight into specific protein conformations. The elbow of the fitting L-curves (indicated by the black arrows) is a solution that simultaneously optimizes both smoothness and peak resolution. Distance distributions shown for each sample are the optimum solution at the L-curve elbow.

ForF-NMR studies, we site-specifically labeled a minimal cysteine version of βAR with a trifluoroacetanilide probe at Cys265, an endogenous residue located at the cytoplasmic end of TM6 ( Figures 1 B and S1 A) (). The resultingF-NMR spectrum displays peaks at different chemical shifts that reflect the unique environments of the trifluoromethyl probe at Cys265 associated with specific conformations of TM6 and neighboring TM3, TM5, and TM7. Each peak defines a given conformation or state. In one-dimensional NMR, the area associated with a peak is in direct proportion to the population of that conformer. The line width reflects subtle conformational heterogeneity or exchange dynamics between states, which can be distinguished by additional Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements (). Thus, NMR spectra reveal conformational heterogeneity in and around TM6, either as peak broadening or as multiple peaks with different chemical shifts. To aid in the visualization ofF-NMR spectra, we subtract a peak originating from another labeled site on the βAR that does not change upon addition of ligands. The original spectrum originating from Cys265 and the subtracted constant peak are shown in Figures S1 B and S1C.

(E) DEER data for β 2 AR single cysteine mutants. Raw data is shown as solid lines with dashed lines indicating the best-fit background function. For both β 2 AR-Δ5-N148C and β 2 AR-Δ5-L266C labeled with IA-PROXYL, the DEER data show no indication of dipolar interaction. Distances for the double cysteine mutant β 2 AR-Δ5-N148C-L266C therefore arise solely from the interaction between nitroxides at 148 in TM4 and 266 in TM6.

(D) Continuous-wave Electron Paramagnetic Resonance (CW-EPR) spectra for β 2 AR-Δ5-N148C and β 2 AR-Δ5-L266C labeled with IA-PROXYL show minimal changes in spin mobility with addition of carazolol, BI167107, or Nb80. Changes in the DEER distribution therefore reflect changes in the conformation of the receptor and not changes in the rotamers of the spin label.

(C) The peak at −61.83 ppm does not change in the presence of agonist, inverse agonist, or Nb80. This peak is due to non-specific labeling of a site with 19 F-BTFA that does not undergo changes in its environment in response to ligands or Nb80.

(B) The spectrum of β 2 AR-Δ4 shows two peaks. The broad peak was assigned to Cys265 using the β 2 AR-Δ5 construct labeled with 19 F-BTFA.

(A) Snake diagram of β 2 AR constructs used in this study. The minimal cysteine β 2 AR-Δ5 construct has the following mutations: C77V, C265A, C327S, C378A, C406A. β 2 AR-Δ4 retains the endogenous cysteine at position 265. For DEER studies, cysteines were introduced at positions 148 (TM4) and 266 (TM6) on the β 2 AR-Δ5 background.

Discussion

2 AR bound to BI167107 and isoproterenol suggest that agonists do not fully stabilize the active conformation of the receptor at the cytoplasmic domain. Furthermore, in each case, agonist-bound β 2 AR is highly dynamic and interconverts between inactive, intermediate, and active conformations with varying timescales. In 2 AR. BI167107 induces a greater decrease in energy of the active-like state S3. Isoproterenol, on the other hand, induces a small decrease in the energy of both the S2 and S3 states. Based upon their unique 19F NMR chemical shifts, we distinguish the active intermediate state S3 stabilized by isoproterenol or BI167107 alone as being distinct from S4, the fully active state stabilized only in the presence of a slowly dissociating agonist and the G-protein mimetic Nb80. It should be noted that we do not observe a difference between S3 and S4 in DEER studies. This may reflect the limits of spatial resolution of DEER spectroscopy or the fact that S3 and S4 have the same maximum distance but differ in the conformation of other TM segments that are near C265, such as TM5 and TM7. Nevertheless, the distinction between S3 and S4 is supported by NMR experiments examining the dynamics of transmembrane 13CH 3 -ε-methionines that revealed the inability of BI167107 to stabilize the transmembrane core of β 2 AR in an active conformation in the absence of Nb80 ( Nygaard et al., 2013 Nygaard R.

Zou Y.

Dror R.O.

Mildorf T.J.

Arlow D.H.

Manglik A.

Pan A.C.

Liu C.W.

Fung J.J.

Bokoch M.P.

et al. The dynamic process of β(2)-adrenergic receptor activation. 2 AR are not tightly allosterically coupled. This “loose allosteric” regulation has previously been proposed based on long timescale molecular dynamics simulations in which agonist-bound β 2 AR in an active conformation rapidly transitions to the inactive state but without a high degree of correlation in conformation between the cytoplasmic domain and the ligand binding pocket ( Dror et al., 2011 Dror R.O.

Arlow D.H.

Maragakis P.

Mildorf T.J.

Pan A.C.

Xu H.

Borhani D.W.

Shaw D.E. Activation mechanism of the β2-adrenergic receptor. Taken together, the spectroscopic results for βAR bound to BI167107 and isoproterenol suggest that agonists do not fully stabilize the active conformation of the receptor at the cytoplasmic domain. Furthermore, in each case, agonist-bound βAR is highly dynamic and interconverts between inactive, intermediate, and active conformations with varying timescales. In Figure 1 D, we illustrate the effects of BI167107 and isoproterenol on βAR. BI167107 induces a greater decrease in energy of the active-like state S3. Isoproterenol, on the other hand, induces a small decrease in the energy of both the S2 and S3 states. Based upon their uniqueF NMR chemical shifts, we distinguish the active intermediate state S3 stabilized by isoproterenol or BI167107 alone as being distinct from S4, the fully active state stabilized only in the presence of a slowly dissociating agonist and the G-protein mimetic Nb80. It should be noted that we do not observe a difference between S3 and S4 in DEER studies. This may reflect the limits of spatial resolution of DEER spectroscopy or the fact that S3 and S4 have the same maximum distance but differ in the conformation of other TM segments that are near C265, such as TM5 and TM7. Nevertheless, the distinction between S3 and S4 is supported by NMR experiments examining the dynamics of transmembraneCH-ε-methionines that revealed the inability of BI167107 to stabilize the transmembrane core of βAR in an active conformation in the absence of Nb80 (). Taken together, these spectroscopic results suggest that the conformation of the ligand-binding pocket and the cytoplasmic domain of βAR are not tightly allosterically coupled. This “loose allosteric” regulation has previously been proposed based on long timescale molecular dynamics simulations in which agonist-bound βAR in an active conformation rapidly transitions to the inactive state but without a high degree of correlation in conformation between the cytoplasmic domain and the ligand binding pocket ().

2 AR by agonists and inverse agonists demonstrated here stands in contrast to what has been observed for the light-sensitive transducer rhodopsin, the GPCR that has been most extensively characterized by biophysical methods ( 2 AR, conformational changes in rhodopsin have previously been studied in a detergent environment. While the dynamic properties of both rhodopsin and β 2 AR are predicted to be influenced by the lipid environment of a cellular membrane, both receptors are functional in dodecyl-maltoside. Using this detergent in the DEER experiments presented here allows us to compare the intrinsic dynamics of the β 2 AR with previously published studies on rhodopsin. In the presence of the covalently attached inverse agonist 11-cis-retinal, EPR and NMR experiments show that TM6 of rhodopsin primarily adopts a single inactive conformation that is partly stabilized by the intracellular ionic lock ( Klein-Seetharaman et al., 1999 Klein-Seetharaman J.

Getmanova E.V.

Loewen M.C.

Reeves P.J.

Khorana H.G. NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution (19)F NMR. Knierim et al., 2007 Knierim B.

Hofmann K.P.

Ernst O.P.

Hubbell W.L. Sequence of late molecular events in the activation of rhodopsin. Smith, 2010 Smith S.O. Structure and activation of the visual pigment rhodopsin. 2 AR is more dynamic in the inactive state, with rapid transitions between conformations likely representing different states of the β 2 AR ionic lock. Figure 6 Differences in the Dynamic Character of Rhodopsin and β 2 AR Show full caption (A) The β 2 AR is conformationally dynamic in the inactive state, and agonists induce further dynamics to varying degree. The active state is only stabilized in the presence of either G protein or a G-protein mimetic. Inverse agonists increase the rate of exchange between ionic lock intact (S1) and broken (S2) states, thereby reducing the lifetime of both states. (B) Dark rhodopsin is minimally dynamic due to the highly efficacious inverse agonist 11-cis-retinal. Illumination by light induces a conformational change to Metarhodopsin II and an accompanying outward displacement of TM6. This active state is then recognized by the G protein transducin (G t ). The regulation of βAR by agonists and inverse agonists demonstrated here stands in contrast to what has been observed for the light-sensitive transducer rhodopsin, the GPCR that has been most extensively characterized by biophysical methods ( Figure 6 ). Similar to the experiments presented here for the βAR, conformational changes in rhodopsin have previously been studied in a detergent environment. While the dynamic properties of both rhodopsin and βAR are predicted to be influenced by the lipid environment of a cellular membrane, both receptors are functional in dodecyl-maltoside. Using this detergent in the DEER experiments presented here allows us to compare the intrinsic dynamics of the βAR with previously published studies on rhodopsin. In the presence of the covalently attached inverse agonist 11-cis-retinal, EPR and NMR experiments show that TM6 of rhodopsin primarily adopts a single inactive conformation that is partly stabilized by the intracellular ionic lock (. In contrast, carazolol-bound βAR is more dynamic in the inactive state, with rapid transitions between conformations likely representing different states of the βAR ionic lock.

s . In addition to the population of active conformations, receptor activation and ligand efficacy may also be governed by the lifetime of inactive states. While the populations of S1 and S2 are not different for unliganded and carazolol-bound receptor ( The structural basis of basal activity and inverse agonism is not known. The spectroscopy studies presented here do not reliably detect a population in the active state for the unliganded receptor. However, given the relatively small population of active intermediate S3 observed in the presence of the full agonist isoproterenol, it is possible that a small amount of S3 that is outside the detection limit of these spectroscopic experiments could result in substantial activation of G. In addition to the population of active conformations, receptor activation and ligand efficacy may also be governed by the lifetime of inactive states. While the populations of S1 and S2 are not different for unliganded and carazolol-bound receptor ( Figure 1 D), we do observe a difference in the exchange rate between these states by CPMG experiments. The more rapid exchange observed in the presence of carazolol and ICI-118,551 is associated with a shorter lifetime of both S1 and S2 ( Figures 1 D and 3 C). In particular, we speculate that the lifetime of S2 (ionic lock broken) is relevant to activation. Conversion between the inactive S2 and active-like S3 states involves a significant energy barrier and a large rearrangement of receptor topology. As such, this conversion process is likely highly sensitive to the lifetime of the ionic lock broken (S2) state. Inverse agonists may reduce the lifetime of S2 below the characteristic timescale required for the conversion from S2 to S3, thereby curtailing receptor activation. Therefore, inverse agonist suppression of basal activity may result from both a decrease in receptor population of intermediate active states and a decreased lifetime of states on the path to activation.