β2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

Short Article

b2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop Graphical Abstract

Authors Matthew T. Eddy, Tatiana Didenko, € thrich Raymond C. Stevens, Kurt Wu

Correspondence [email protected]

In Brief G protein-coupled receptors (GPCRs) exist in function-related equilibria of locally different conformational states. Eddy, Didenko et al. show how fusion of a soluble protein into the third intracellular loop, as is widely used in crystallographic studies of GPCRs, modifies these equilibria.

Highlights d

d

d

19

F-NMR conformational studies of GPCR fusion protein used for crystallography

Nearly full population of b2AR active-like state without intracellular protein bound Different response to ligand efficacy for G-protein and arrestin signaling

Eddy et al., 2016, Structure 24, 1–8 December 6, 2016 ª 2016 Elsevier Ltd. http://dx.doi.org/10.1016/j.str.2016.09.015

Please cite this article in press as: Eddy et al., b2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.015

Structure

Short Article b2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop € thrich2,3,5,* Matthew T. Eddy,1,4 Tatiana Didenko,2,4 Raymond C. Stevens,1 and Kurt Wu 1Departments of Biological Sciences and Chemistry, Bridge Institute, The University of Southern California, South Vermont Avenue, MC 3303, Los Angeles, CA 90089, USA 2Department of Integrative Structural and Computational Biology 3Skaggs Institute of Chemical Biology The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA 4Co-first author 5Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2016.09.015

SUMMARY

Fluorine-19 nuclear magnetic resonance (NMR) was used to study conformational equilibria at the intracellular tips of helices VI and VII in a variant b2-adrenergic receptor (b2AR) containing T4-lysozyme fused into the third intracellular loop (b2AR-T4L), a G protein-coupled receptor (GPCR) modification widely used in crystal structure determination. G-protein signaling at helix VI showed nearly complete population of an active-like state for all ligand efficacies in the absence of an intracellular protein. For arrestin signaling at helix VII, a native-like equilibrium was observed, except for complexes with ligands devoid of a hydrophobic moiety at the ethanolamine end. These data confirm that response of G-protein and arrestin signaling to ligand efficacy is not coupled, and presents evidence for long-range effects between fusion protein and orthosteric binding cavity, which are suppressed by voluminous bound ligands. Solution NMR thus provides complementary information, which should be considered in functional interpretations of GPCR crystal structures obtained with ICL3 fusions.

INTRODUCTION Much of our understanding of structure-function relationships of G protein-coupled receptors (GPCRs) comes from the determination of their three-dimensional structures by X-ray crystallography, as represented today by an extensive database of GPCR crystal structures (www.rcsb.org; see also Table 1). A widely used technique to crystallize GPCRs is the insertion of a soluble protein into the third intracellular loop (ICL3) of the receptor. This method was originally introduced to facilitate crystallization of the membrane protein lactose permease from Escherichia coli (Engel et al., 2002; Prive et al., 1994). It was then successfully applied to crystallize the human b2-adrenergic receptor (b2AR), where the ICL3 was modified by fusion with the soluble protein

T4-lysozyme to yield b2AR-T4L (Cherezov et al., 2007; Rosenbaum et al., 2007). To validate this approach, studies of b2AR-T4L with fluorescence spectroscopy were interpreted as showing that the ICL3 fusion had at most minor effects on function-related conformational equilibria (Rosenbaum et al., 2007). Based largely on these data, the method was then extensively used to facilitate GPCR crystallization, accounting presently for 23 out of the 33 published unique GPCR crystal structures, and 46 structures of these GPCRs in complexes with additional, different ligands (Table 1). For two GPCRs, b2AR and the A2A adenosine receptor (A2AAR), crystal structures have been obtained with (Cherezov et al., 2007; Jaakola et al., 2008; Rosenbaum et al., 2007) and without (Dore´ et al., 2011; Zou et al., 2012) T4L fusions in ICL3. Whereas the data for b2AR with and without the fusion protein are of widely different quality, preventing detailed interpretation, the A2AAR structures show a small displacement of helix VI between the two structures. In this paper, we describe new comparative studies of b2AR and b2AR-T4L using 19F-nuclear magnetic resonance (NMR) spectroscopy in solution. Spectroscopic studies of GPCRs in solution, especially NMR spectroscopy, complement GPCR crystallography by identifying multiple, simultaneously populated, conformational states in function-related equilibria. Such data provide deepened insight into the mechanisms of GPCR signal transduction, as described in detail, for example, in studies of the b2AR (Bokoch et al., 2010; Chung et al., 2012; Didenko et al., 2013; Horst et al., 2012, 2013; Kim et al., 2013; Kofuku et al., 2014; Liu et al., 2012a; Nygaard et al., 2013; Sounier et al., 2015), the m-opioid receptor (Okude et al., 2015; Sounier et al., 2015), the turkey b1-adrenergic receptor (Isogai et al., 2016), and the A2AAR (Ye et al., 2016). Extension of such studies to b2ART4L seemed to be of special interest, considering the extensive database of crystal structures determined with the ICL3 fusion approach (Table 1). RESULTS NMR studies of b2AR, where 2,2,2-trifluoroethanethiol (TET) 19F labels were introduced at the cytoplasmic ends of the helices VI (C265), VII (C327), and VIII (C341), had previously revealed function-related local conformational equilibria (Liu et al., 2012a), Structure 24, 1–8, December 6, 2016 ª 2016 Elsevier Ltd. 1

Please cite this article in press as: Eddy et al., b2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.015

Table 1. GPCR Crystal Structures Determined with an ICL3 Fusion Protein Receptor

PDB ID

Fusion Protein

Resolution (A˚)

Ligand

TM V Junctiona

TM VI Junctiona

Reference

A2A adenosine receptor

4EIY

BRIL

1.80

ZM241385

5.70 ( 5)

6.20 ( 1)

Liu et al., 2012b

A2A adenosine receptor

3QAK

T4L

2.71

UK432097

5.70 ( 5)

6.23 (+3)

Xu et al., 2011

A2A adenosine receptor

3EML

T4L

2.60

ZM241385

5.70 ( 5)

6.23 (+3)

Jaakola et al., 2008

b2-Adrenergic receptor

2RH1

T4L

2.40

carazolol

5.73 ( 3)

6.21 ( 3)

Cherezov et al., 2007

b2-Adrenergic receptor

3D4S

T4L

2.80

timolol

5.73 ( 3)

6.21 ( 3)

Hanson et al., 2008

b2-Adrenergic receptor

3NY8

T4L

2.84

ICI 118,551

5.73 ( 3)

6.21 ( 3)

Wacker et al., 2010

b2-Adrenergic receptor

3NY9

T4L

2.84

inverse agonistb

5.73 ( 3)

6.21 ( 3)

Wacker et al., 2010

b2-Adrenergic receptor

3NYA

T4L

3.16

alprenolol

5.73 ( 3)

6.21 ( 3)

Wacker et al., 2010

b2-Adrenergic receptor

3P0G

T4L

3.50

BI-167107

5.73 ( 3)

6.21 ( 3)

Rasmussen et al., 2010

b2-Adrenergic receptor

3PDS

T4L

3.50

FAUC50

5.73 ( 3)

6.21 ( 3)

Rosenbaum et al., 2010

d-Opioid receptor

4EJ4

T4L

3.40

naltrindol

5.69 (+1)

6.20 (+3)

Granier et al., 2012

k-Opioid receptor

4DJH

T4L

2.90

JDTic

5.73 (+5)

6.24 (+1)

Wu et al., 2012

m-Opioid receptor

4DKL

T4L

2.80

b-funaltrexamine

5.68 (0)

6.22 ( 1)

Manglik et al., 2012

OX1 orexin receptor

4ZJ8

PGS

2.75

suvorexant

5.70 (+1)

6.24 ( 5)

Yin et al., 2016

OX1 orexin receptor

4ZJC

PGS

2.83

SB-674042

5.70 (+1)

6.24 ( 5)

Yin et al., 2016

OX2 orexin receptor

4S0V

PGS

2.50

suvorexant

5.70 (+1)

6.24 ( 5)

Yin et al., 2015

M1 muscarinic acetylcholine receptor

5CXV

T4L

2.70

tiotropium

5.69 ( 6)

6.24 (0)

Thal et al., 2016

M2 muscarinic acetylcholine receptor

3UON

T4L

3.00

3-quinuclidinylbenzilate

5.70 ( 7)

6.24 (0)

Haga et al., 2012

M3 muscarinic acetylcholine receptor

4DAJ

T4L

3.40

tiotropium

5.67 ( 9)

6.25 (+2)

Kruse et al., 2012

M4 muscarinic acetylcholine receptor

5DSG

T4L

2.60

tiotropium

5.69 ( 5)

6.24 (0)

Thal et al., 2016

NTS1 neurotensin receptor

4GRV

T4L

2.80

neurotensin peptide

5.71 (0)

6.31 (+8)

White et al., 2012

NTS1 neurotensin receptor

4BUO

T4L

2.75

neurotensin peptide

5.71 (0)

6.31 (+8)

Egloff et al., 2014

NTS1 neurotensin receptor

4XES

T4L

2.60

neurotensin peptide

5.71 (0)

6.31 (+8)

Krumm et al., 2015

Dopamine D3 receptor

3PBL

T4L

2.89

eticlopride

5.71 (+5)

6.25 ( 1)

Chien et al., 2010

Histamine H1 receptor

3RZE

T4L

3.10

doxepin

5.70 ( 5)

6.24 (0)

Shimamura et al., 2011

P2Y12 receptor

4NTJ

BRIL

2.62

AZD1283

5.72 (+4)

6.23 ( 6)

Zhang et al., 2014b

P2Y12 receptor

4PXZ

BRIL

2.50

2MeSADP

5.72 (+4)

6.23 ( 6)

Zhang et al., 2014a

P2Y12 receptor

4PY0

BRIL

3.10

2MeSATP

5.72 (+4)

6.23 ( 6)

Zhang et al., 2014a

Sphingosine 1-phosphate receptor

3V2W

T4L

3.35

sphingolipid mimic

5.69 (+1)

6.22 ( 2)

Hanson et al., 2012 c

Sphingosine 1-phosphate receptor

3V2Y

T4L

2.80

sphingolipid mimic

5.69 (+1)

6.22 ( 2)

Hanson et al., 2012

LPA1 Lysophosphatidic Acid Receptor 1

4Z34

BRIL

3.00

ONO-9780307

5.65 ( 1)

6.25 (+4)

Chrencik et al., 2015

LPA1 Lysophosphatidic Acid Receptor 1

4Z35

BRIL

2.90

ONO-9910539

5.65 ( 1)

6.25 (+4)

Chrencik et al., 2015

LPA1 Lysophosphatidic Acid Receptor 1

4Z36

BRIL

2.90

ONO-3080573

5.65 ( 1)

6.25 (+4)

Chrencik et al., 2015

5-HT1B serotonin receptor

4IAR

BRIL

2.70

ergotamine

5.70 ( 7)

6.26 (+3)

Wang et al., 2013

5-HT1B serotonin receptor

4IAQ

BRIL

2.80

dihydroergotamine

5.70 ( 7)

6.24 (+1)

Wang et al., 2013

5-HT2B serotonin receptor

4IB4

BRIL

2.70

ergotamine

5.70 ( 7)

6.24 (0)

Wacker et al., 2013

5-HT2B serotonin receptor

4NC3

BRIL

2.80

ergotamine

5.70 ( 7)

6.24 (0)

Liu et al., 2013 d

Protease-activated receptor 1 (PAR1)

3VW7

T4L

2.20

vorapaxar

5.69 (+2)

6.25 ( 2)

Zhang et al., 2012

CXCR4 chemokine receptor

3ODU

T4L

2.50

IT1t

5.68 (+1)

6.26 ( 4)

Wu et al., 2010 e

CXCR4 chemokine receptor

3OE8

T4L

3.10

IT1t

5.68 (+1)

6.26 ( 4)

Wu et al., 2010 e

CXCR4 chemokine receptor

3OE9

T4L

3.10

IT1t

5.68 (+1)

6.26 ( 4)

Wu et al., 2010 e (Continued on next page)

2 Structure 24, 1–8, December 6, 2016

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Table 1.

Continued

Receptor

PDB ID

Fusion Protein

Resolution (A˚)

Ligand

TM V Junctiona

TM VI Junctiona

Reference

CXCR4 chemokine receptor

3OE6

T4L

3.20

IT1t

5.68 (+1)

6.26 ( 4)

Wu et al., 2010 e

CXCR4 chemokine receptor

3OE0

T4L

2.90

CVX15

5.68 (+1)

6.26 ( 4)

Wu et al., 2010 e

CXCR4 chemokine receptor

4RWS

T4L

3.10

vMIP-II

5.68 (+1)

6.26 ( 4)

Burg et al., 2015

CCR5 chemokine receptor

4MBS

rubredoxin

2.71

maraviroc

5.68 (+1)

6.26 ( 3)

Tan et al., 2013

GPR40 free fatty acid receptor 1 (FFAR1)

4PHU

T4L

2.33

TAK-875

5.69 (+2)

6.25 ( 3)

Srivastava et al., 2014

a

Numbers are in the Ballesteros-Weinstein numbering system (Ballesteros and Weinstein, 1995); for residues located in the loops, the BallesterosWeinstein number is relative to the last residue in helix V or VI. In addition, in parentheses the positions of fusion protein insertions relative to the intracellular tips of helices V and VI are indicated, as determined from the GPCRDB (www.gpcrdb.org), which uses a structure-based sequence alignment (Munk et al., 2016). Negative numbers indicate positions closer to the N terminus and positive numbers indicate positions closer to the C terminus, i.e., for helix V, negative numbers indicate closer contact with the intracellular tip, and for helix VI positive numbers indicate closer contact with the intracellular tip. b Compound selected as described in Kolb et al. (2009). c Data processed with experimental microdiffraction data assembly method. d Diffraction data obtained from X-ray free electron laser source (XFEL). e These are different structures of the same ligand-receptor complex crystallized in different space groups.

with exchange rates between the limiting states slower than 10 s 1 (Horst et al., 2013). Single-cysteine variants of b2AR were studied in complexes with different ligands, exhibiting variable pharmacological efficacies, and without ligand added to the sample. Based on these data, chemical shifts of exactly one ‘‘inactive’’ and one ‘‘active-like’’ conformational state were assigned at each of the two 19F-NMR probes at the positions 265 and 327. In the present study we repeated these measurements with b2AR-T4L, which contained T4-lysozyme fused into the ICL3 between helices V and VI, and 19F-NMR probes at the same positions as in the previous study of b2AR (Figure 1A). We then analyzed the differences between b2AR and b2AR-T4L. The 19F probe located at the tip of helix VIII served as an internal control for the b2AR-T4L experiments, because spectra of b2ART4L(C265A, C327S, TETC341) were not responsive to the presence of different ligands, as was also observed for the same probe position in b2AR (Liu et al., 2012a). In b2AR-T4L the Local Conformational Equilibrium at Helix VI Is Shifted Toward the Active-like State, Irrespective of the Orthosteric Ligand Bound The 19F-NMR spectra measured at 280 K of all b2AR-T4L (TETC265, C327S, C341A) complexes in the present study exhibited only a single signal, which contrasts with the twocomponent resonances observed for b2AR. There is thus no relation with the efficacy of the bound orthosteric ligand. Within the precision of our measurements the chemical-shift value of the single component was identical to the value that had previously been assigned to the active-like state of b2AR(TETC265, C327S, C341A) (Figure 1B). This shift toward the active-like state at helix VI is robust, as it is maintained over the temperature range from 280 K to 310 K (Figure S1), which contrasts with the temperature-induced population shift detected in b2AR (TETC265, C327S, C341A) (Horst et al., 2013; Liu et al., 2012a). The absence of a detectable response at helix VI to variable efficacies of ligands bound to the orthosteric cavity can be rationalized by a situation where the populations of all conformations

other than the active-like state are too small for their NMR signals to be observed. Effect of T4L Fusion into ICL3 on the Conformational Equilibria of Helix VII In contrast to the observations at the 19F-NMR probe at position 265, the 19F-NMR spectra of b2AR-T4L at C327, located at the cytoplasmic tip of helix VII, showed two distinct components for all complexes, representing two locally different conformational states (Figure 2). This observation shows that, in contrast to the data obtained for helix VI, the conformational equilibrium at helix VII is less profoundly perturbed by the introduction of the fusion protein into the ICL3. For a more quantitative comparison of the helix VII equilibria recorded for b2AR-T4L and b2AR, we deconvoluted the 19 F-NMR data obtained for b2AR-T4L(C265A, TETC327, C341A) with a double-Lorentzian function, to estimate the relative populations of active-like and inactive states. Firstly, this showed that the chemical shifts and widths of the NMR peaks are identical to those that were previously assigned to the active-like and inactive states of b2AR(C265A, TETC327, C341A) (Liu et al., 2012a). Therefore, we could directly compare the corresponding relative peak volumes (Figure 2). This showed a close association between b2AR and b2AR-T4L, except that we observed stronger shifts of the equilibrium toward the active-like state for b2ART4L in the complex with norepinephrine, and for the apo-form obtained with no ligand added. Norepinephrine is the smallest ligand used in the present study, lacking an acyl group at the ethanolamine end (Figure 2), and for the sample with no ligand added (Apo), the binding cavity is expected to contain only even smaller molecules. These apparent outliers thus seem to be related with the size of the ligand bound in the orthosteric site, as discussed in more detail below (Figure 3). DISCUSSION The present NMR studies of function-related conformational equilibria of b2AR and the variant b2AR-T4L, which has been Structure 24, 1–8, December 6, 2016 3

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A

C265 C327 C341

β2AR (TETC265)

β2AR–T4L (TETC265)

AI

B

A I

Carazolol

Alprenolol

Apo

Clenbuterol

Isoetharine

Norepinephrine

Formoterol

12

10

8

12

10

δ(19F) [ppm]

Figure 1. Locations of the 19F-NMR Probes in b2AR-T4L Used in This Work and Ligand-Efficacy-Dependent Effects on Local Conformational Equilibria Observed at C265 (A) Locations of TET 19F-NMR labels in the crystal structure of b2AR-T4L in complex with carazolol (PDB: 2RH1) (Cherezov et al., 2007). b2AR and T4L are shown as gray ribbon and blue surface representations, respectively. TETlabeled cysteines at positions 265, 327, and 341 are indicated as yellow spheres. (B) 1D 19F-NMR spectra of b2AR-T4L(TETC265, C327S, C341A) and b2AR (TETC265, C327S, C341A) measured at 280 K for complexes with six different ligands and without addition of a ligand. The data for b2AR (TETC265, C327S, C341A) were taken from Liu et al. (2012a). Dashed vertical red and blue lines indicate the previously assigned chemical shifts (Liu et al., 2012a) of the active-

4 Structure 24, 1–8, December 6, 2016

essential for success with GPCR crystal structure determination, provide information that will need to be considered in functional interpretations of crystal structure data obtained with variant GPCRs containing fusion proteins in ICL3. Novel insights into function-related conformational equilibria in b2AR (Figure 3) were obtained using two TET 19F-NMR probes located at the intracellular tips of the transmembrane helices VI and VII to measure the impact of fusing T4L into the ICL3. This allowed us to obtain information on two distinct pathways, signaling, respectively, to G protein (Rasmussen et al., 2011) and to b-arrestin (Kang et al., 2015; Nobles et al., 2011). For the G-protein signaling pathway, T4L in ICL3 caused a shift of the conformational equilibrium toward the active-like state, eliminating the previously characterized relation with the efficacy of bound orthosteric ligands (Liu et al., 2012a). For the arrestin pathway, we observed a long-range effect of T4L for complexes with small ligands, whereas for ligands with hydrophobic substituents at the ethanolamine end (Figure 2) the relation with ligand efficacy was preserved. The qualitatively different behavior of the arrestin and G-protein signaling pathways confirmed that the response of the two pathways to orthosteric ligands is not coupled (Liu et al., 2012a). As the ‘‘linker’’ between T4L or other proteins and the intracellular tip of helix VI in the 23 different GPCR crystal structures obtained with ICL3 fusions (Table 1) varies between one and five residues, and is five residues for b2AR-T4L, the present observations are likely to represent a general response of GPCRs to ICL3 fusions. Specific Individual Labeling of Two Different Residues Provides New Insights into the Impact of the T4L Fusion in ICL3 on Function-Related Conformational Equilibria The effects of fusing T4-lysozyme into ICL3 of b2AR were examined previously by comparing fluorescence emission spectra of bimane-labeled b2AR-T4L and b2AR complexes with antagonists and agonists (Rosenbaum et al., 2007), which showed only minor effects of the T4L on the fluorescence response; at the time, this was interpreted as documenting absence of an influence of T4L on the conformational behavior at the position of C265. Our data now show that there are no measurable ligandrelated variations of the conformational equilibrium at C265 of b2AR-T4L. This apparent discrepancy can be explained by the different availability of cysteine groups for labeling with sulfhydryl-reactive reagents in the two experiments. From experience with 19F-NMR studies of TET-labeled b2AR (Horst et al., 2013; Liu et al., 2012a) and with single-molecule fluorescence studies of Cy3-labeled b2AR (Lamichhane et al., 2015), it is now known that the three cysteines C265, C327, and C341 all react with the labeling reagents. Therefore, single-cysteine variants of b2AR were used here and in our earlier work (Liu et al., 2012a) to obtain spectra from individually labeled cysteines (Figures 1 and 2). In the earlier study of b2AR-T4L with bimane fluorescence

like and inactive functional states for b2AR (TETC265, C327S, C341A), and NMR signals representing these states are shown in red (active-like) and blue (inactive). All experiments were measured at 280 K, and the following parameters were used to measure and process the spectra: data size 1,024 complex points, acquisition time 51 ms, 24,576 scans per experiment. Prior to Fourier transformation the data were multiplied with an exponential function with a line-broadening factor of 30 Hz and zero-filled to 2,048 points.

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β2AR (TETC327)

β2AR–T4L (TETC327)

I A

Figure 2. Helix VII Ligand-Efficacy-Related Conformational Equilibrium Is Preserved in b2AR-T4L Complexes Except for the Norepinephrine Complex

I A Carazolol

1D 19F-NMR spectra of b2AR-T4L(C265A, TETC327, C341A) and b2AR(C265A, TETC327, C341A) measured with six ligands and without addition of a ligand (‘‘Apo’’) are shown, as recorded at 280 K. The data for b2AR(C265A, TETC327, C341A) were taken from Liu et al. (2012a). Deconvolution with Lorentzian line shapes representing individual components are indicated in blue (inactive state) and red (active-like state). Dashed red and blue vertical lines correspond to the previously assigned chemical shifts for the active-like and inactive functional states for b2AR(C265A, TETC327, C341A) (Liu et al., 2012a). Thin black and thick gray lines indicate the experimental data and the fitting results, respectively. Chemical structures of the ligands used in the current work are displayed on the right, where the ethanolamine substituents are highlighted in yellow.

HN

O

N + OH H 2

O

N + OH H 2

Alprenolol

Apo

No Ligand Added Cl H 2N

Clenbuterol

Cl

N + OH H 2

due to the use of chemically different 19FNMR probes and different detergents. Specifically, it could be that the presumed rapidly exchanging multiple states reported in the aforementioned studies with ternary complexes would give rise to single merged signals in our experiments.

HO

Isoetharine

HO

Norepinephrine

N + OH H 2

HO NH 3+

HO OH

Formoterol

HO O

12

10

8

12

10

HN

(19F) [ppm]

spectroscopy (Rosenbaum et al., 2007), labeling was performed with all three reactive cysteines present. We therefore conclude that the observed fluorescence response must have been due to the chromophore attached to C327 on helix VII, which, based on the current work, retains a native-like conformational equilibrium in the complexes used in the study of Rosenbaum et al. (2007) (Figures 2 and 3), rather than to a response at C265. No New Conformational States Were Observed for b2ART4L at C265 and C327 In the context with literature data (see below), the following observation is intriguing: although in b2AR-T4L the equilibrium at helix VI is shifted essentially completely toward the activelike state in the absence of a bound intracellular protein, only the previously assigned inactive and active-like, locally different b2AR states, as characterized by their 19F-NMR shifts, were observed at the positions of both C265 and C327. Our data are in contrast with interpretations of 19F-NMR data on b2AR labeled at C265 in ternary complexes with ligands and nanobodies (Chung et al., 2012; Kim et al., 2013; Staus et al., 2016), which were analyzed as showing evidence for hidden signals of additional states. These apparent discrepancies may be

Impact of T4L in ICL3 on Arrestin Signaling Pathway Is Suppressed in Complexes with Larger Ligands N + A long-range influence of the T4L fusion OH H 2 into ICL3 on the arrestin signaling pathway results in increased population of the active-like state at helix VII of b2AR-T4L with no ligand added (Apo), and for the complex with norepinephrine. These two preparations contain smaller moieties in the orthosteric binding cavity than the other complexes included in this study (Figures 2 and 3), for which there was no measurable effect from T4L. A likely rationale for these observations comes from earlier work. The structure of b2ART4L in complex with carazolol (Cherezov et al., 2007) revealed hydrogen bond formation between the ethanolamine end of carazolol and the side chains of D113 and N312, forming a polar network between the ligand and receptor. Molecular modeling predicted that these interactions are preserved for b2AR complexes with agonists (Katritch et al., 2009), and biochemical studies showed that replacement of N312 with alanine resulted in a 100-fold reduction of binding affinity for both antagonists and agonists (Suryanarayana and Kobilka, 1993), supporting the importance of this interaction. Ligand binding assays also showed that b2AR-T4L has increased affinity for agonist binding when compared with b2AR (Rosenbaum et al., 2007). In apob2AR-T4L and the complex with norepinephrine, the observed long-range influence of T4L in ICL3 on the 19F label at the tip of helix VII may be facilitated by weakening or absence of the hydrogen bonding network, which, in concert with the small O

Structure 24, 1–8, December 6, 2016 5

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A

2

AR

B

2

AR–T4L

C

2

AR–T4L IV

III

Figure 3. Visualizing the Effects of T4L on Local Conformational Equilibria Related to the G-Protein and Arrestin Signaling Pathways in b2AR-T4L

(A) Schematic side-view representation of b2AR. The transmembrane helices I to V are shaded in V VII gray. The helices VI and VII, which are in equilibrium VII II VI between two conformational states manifested by T4L VI different NMR chemical shifts of 19F probes C327 VII I attached to C265 and C327 (Liu et al., 2012a), are C265 represented as dashed rectangles in blue and T4L cyan, respectively. The signaling pathways to G VII VII VI protein (blue) and b-arrestin (cyan) are indicated, and the positions of the 19F-NMR labels are shown C327 by yellow spheres, where the active-like state is IV III Carazolol identified by a black circle. The orthosteric ligand Alprenolol C265 Clenbuterol binding cavity is indicated by green shading. For all V VII Isoetharine VI II ligands listed in (B), the response of the local Formoterol T4L conformational equilibria at the intracellular tips of G Protein -arrestin VI C327 helices VI and VII in b2AR is related with the efficacy VII I of the ligand bound, i.e., the population of the C265 active-like state is higher for ligands with higher T4L efficacy (Liu et al., 2012a; see Figures 1 and 2). (B) Schematic side views of b2AR-T4L. Same presentation as in (A), except that helix VI is represented by solid blue lines, the T4L fusion protein is depicted as a red oval, and its impact on local conformational equilibria is indicated by red arrows and the intensity of red shading. (C) View from the extracellular surface of the b2AR-T4L complexes shown in (B). Circles indicate the helices I to VII. Ligands are represented in green, with black rectangles indicating hydrogen bonds between the ligand, N113 in helix III, and N312 in helix VII. In the upper panel, red arrows indicate that long-range effects from T4L cause a contraction of the ligand binding pocket (see text). Overall, (B) and (C) illustrate that the T4L fusion has a strong effect on the adjacent helix VI, and that a long-range effect via the orthosteric ligand binding cavity on the 19F-NMR probe at the tip of helix VII is suppressed in the presence of ligands with hydrophobic substituents at the ethanolamine end (Figure 2) (see text). Apo-form Norepinephrine

size of the ligands, could cause a contraction of the orthosteric ligand binding pocket (Figure 3). Intriguingly, ligand binding assays for A2AAR with T4L fused into ICL3 (A2AAR-T4L) showed increased affinity for agonist binding when compared with A2AAR (Jaakola et al., 2008), suggesting that other GPCRs may also show long-range interactions transmitted by contraction of the orthosteric binding pocket in response to ICL3 fusion proteins. In conclusion, comparison of b2AR-T4L with b2AR revealed important differences in the function-related local conformational equilibria of this variant protein, which has been widely used in crystal structure determinations of GPCRs (Table 1). Most important, evidence was obtained for a novel long-range effect from the intracellular end of helix VI through the orthosteric ligand binding cavity to the intracellular tip of helix VII. This is particularly intriguing in view of the observation by Carpenter et al. (2016) that binding of an intracellular partner protein near the tip of helix VI in an A2A adenosine receptor-mini-Gs complex did not result in observable changes at the orthosteric ligand binding site. On the other hand, part of the aforementioned long-range interaction observed here, from T4L at the tip of helix VI to the orthosteric ligand binding site, appears to be in line with recent observations of ‘‘allosteric effects’’ (Staus et al., 2016) on ligand affinity through binding of G protein or substitute proteins in the G-protein binding site near the intracellular tip of helix VI (DeVree et al., 2016).

The b2AR-T4L protein used in this study included the thermally stabilizing mutation E122W (Roth et al., 2008), C-terminal truncation at residue 348, an FLAG tag at the N terminus, and a 63 His tag at the C terminus. b2AR constructs from previous 19F-NMR studies (Horst et al., 2013; Liu et al., 2012a), which were used for comparison in this study, included the same features. Following the previously described construct design for b2AR-T4L (Rosenbaum et al., 2007), residues 231 to 262 in ICL3 (connecting helices V and VI) were replaced by residues 2 to 164 of the T4L protein. Expression, purification, and labeling of b2AR-T4L samples with TET followed published protocols (Liu et al., 2012a). The final NMR buffer was the same for all b2AR-T4L and b2AR samples, and the concentration of ligands was the same for corresponding receptor-ligand complexes of b2AR-T4L and b2AR: carazolol (Toronto Research Chemicals) was 50 mM; alprenolol (Sigma), clenbuterol (Toronto Research Chemicals), norepinephrine (Sigma), isoetharine (Sigma), and formoterol (Toronto Research Chemicals) were 1 mM each. See Supplemental Experimental Procedures for more details. 19 F-NMR spectra were measured on a Bruker AVANCE spectrometer operating at 600 MHz 1H frequency and equipped with a QCI 1H/19F-13C/15N quadruple resonance cryoprobe with a shielded z-gradient coil. The sample temperature was measured with a standard calibration sample of 4% methanol in D4-MeOH. 19F chemical shifts were calibrated to an internal trifluoroacetic acid standard. Acquisition and processing parameters were identical for all b2AR and b2AR-T4L samples (see Supplemental Experimental Procedures for details). SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and one figure and can be found with this article online at http://dx.doi.org/ 10.1016/j.str.2016.09.015. AUTHOR CONTRIBUTIONS

EXPERIMENTAL PROCEDURES PCR site-directed mutagenesis (QuikChange; Stratagene) was used to generate b2AR-T4L cysteine mutants, and primers for site-directed mutagenesis were obtained from Integrated DNA Technology.

6 Structure 24, 1–8, December 6, 2016

M.T.E. performed protein purification and sample characterization. M.T.E. and T.D. recorded NMR data, and M.T.E., T.D., and K.W. analyzed NMR data. R.C.S. and K.W. designed the study. R.C.S. provided input to M.T.E., T.D., and K.W., who wrote the manuscript.

Please cite this article in press as: Eddy et al., b2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop, Structure (2016), http://dx.doi.org/10.1016/j.str.2016.09.015

ACKNOWLEDGMENTS The authors acknowledge support from NIH/NIGMS PSI: Biology grant U54 GM094618. M.T.E. acknowledges funding from an American Cancer Society postdoctoral fellowship. The authors also wish to thank Dr. Jeff Liu, Dr. Reto Horst, and Dr. Vsevolod Katritch for helpful discussions; Dr. Meihua Chu and Tam Trinh for production of b2AR–TL biomass; Jeffrey Velasquez for cloning b2AR–T4L constructs; and Yekaterina Kadyshevskaya for help with preparation of figures. The authors also thank Angela Walker for careful checking of the manuscript.

Engel, C.K., Chen, L., and Prive´, G.G. (2002). Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease. Biochim. Biophys. Acta 1564, 38–46. Granier, S., Manglik, A., Kruse, A.C., Kobilka, T.S., Thian, F.S., Weis, W.I., and Kobilka, B.K. (2012). Structure of the d-opioid receptor bound to naltrindole. Nature 485, 400–404. Haga, K., Kruse, A.C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W.I., Okada, T., Kobilka, B.K., Haga, T., et al. (2012). Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 23–28.

Received: August 3, 2016 Revised: September 16, 2016 Accepted: October 7, 2016 Published: November 10, 2016

Hanson, M.A., Cherezov, V., Griffith, M.T., Roth, C.B., Jaakola, V.-P., Chien, E.Y.T., Velasquez, J., Kuhn, P., and Stevens, R.C. (2008). A specific cholesterol binding site is established by the 2.8 A˚ structure of the human b2-adrenergic receptor. Structure 16, 897–905.

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