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Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.
Muscarinic acetylcholine receptors (M1 – M5) are GPCRs that regulate the activity of a diverse array of central and peripheral functions in the human body, including the parasympathetic actions of acetylcholine1. The M2 muscarinic receptor subtype plays a key role in modulating cardiac function and many important central processes, such as cognition and pain perception1. As it was among the first GPCRs to be purified2 and cloned3, the M2 receptor has long served as a model system in GPCR biology and pharmacology. Muscarinic receptors have attracted particular interest due to their ability to bind small molecule allosteric modulators4. Since allosteric sites are often less conserved than the orthosteric binding site, some ligands binding to allosteric sites show substantial subtype selectivity5,6. Such agents hold great promise for the development of drugs for the treatment of conditions, including diseases of the central nervous system and metabolic disorders. Though crystal structures were recently obtained for inactive states of the M2 and M3 muscarinic receptors7,8, there are no structures of a GPCR bound to an allosteric modulator.
The binding of an agonist to the extracellular side of a GPCR results in conformational changes that enable the receptor to activate heterotrimeric G proteins. Despite the importance of this process, only the β2AR and rhodopsin have been crystallized in fully active conformations9-13. Crystallization of active-state GPCRs has been challenging due to their inherent conformational flexibility and biochemical instability14. To better understand the mechanistic details underlying GPCR activation and allosteric modulation, we solved X-ray crystal structures of the M2 receptor bound to the high affinity agonist iperoxo15 alone and in combination with LY2119620, a positive allosteric modulator.
Initial crystallization attempts with M2 receptor bound to agonists were unsuccessful, likely due to the flexibility of the intracellular receptor surface in the absence of a stabilizing protein. We thus sought to obtain a ‘G protein mimetic’ nanobody for the M2 receptor to facilitate crystallization of the β2AR in an active conformation11. Llamas were immunized with M2 receptor bound to the agonist iperoxo, and a post-immune single variable domain (VHH) nanobody cDNA library was constructed and displayed on the surface of yeast (Fig. 1a).
An essential component for the selection of active-state stabilizing nanobodies was simultaneous staining of yeast with both agonist and inverse-agonist occupied M2 receptor populations, which were distinguishably labeled with separate fluorophores. This allowed the use of fluorescence-activated cell sorting (FACS) to select those clones binding only agonist-occupied receptor (Fig. 1b; see Online Methods). To ensure that the different fluorophore-conjugated receptors represent distinct receptor populations requires that at least one receptor population must be bound to an exceptionally high-affinity or irreversible ligand. We therefore developed a covalent muscarinic receptor agonist for use in selection experiments. This has precedent in an acetylcholine mustard16, which is thought to react with the binding site residue Asp1033.32 to form a covalent adduct17. Accordingly, we synthesized an analogous “iperoxo mustard,” which we call FAUC123 (Supplementary Methods). We found that FAUC123 bound covalently and was able to induce activation of the M2 receptor (Extended Data Fig. 1), thereby allowing simultaneous staining of yeast with agonist- and antagonist-bound M2 receptor labeled with distinct fluorophores for each population.
After nine rounds of conformational selection, almost all remaining yeast cell clones preferentially bound FAUC123-occupied receptor (Fig. 1d). Three clones in particular, Nb9-1, Nb9-8, and Nb9-20 (Fig. 2a; see Online Methods) exhibited strong, conformationally-selective staining on yeast (Fig. 2b). All three nanobodies enhanced agonist affinity (Fig. 2c), indicating that they stabilize active states of the receptor. Nb9-8 was the most potent, with an EC50 of approximately 100 nM. At high concentrations, Nb9-8 enhanced the affinity of the M2 receptor for iperoxo to almost the same extent as that observed in the presence of the heterotrimeric G protein Gi (Fig. 2d).
M2 receptor was purified in the presence of 10 μM iperoxo, and we obtained crystals of iperoxo-bound M2 receptor in complex with Nb9-8 by lipidic mesophase crystallography. The structure was solved by microdiffraction at Advanced Photon Source beamline 23ID-D (Extended Data Table 1). Supplementing the optimized crystallization conditions with the positive allosteric modulator LY2119620 yielded crystals of M2 receptor simultaneously bound to both iperoxo and the modulator (see Online Methods). For all crystallization work, the agonist iperoxo was used rather than acetylcholine, as the latter is of lower affinity and is prone to hydrolysis.
A key feature of GPCR activation is an outward movement of the intracellular portion of transmembrane (TM) helices 5 and 6, creating a cavity large enough to accommodate the carboxy terminus of the G protein α subunit10,13. While several GPCRs have been crystallized in complex with agonists, only the β2AR and rhodopsin show a fully active state with adequate space to allow G protein binding (Extended Data Fig. 2). As anticipated based on functional studies (Fig. 2), Nb9-8 binds to the intracellular surface of the receptor (Fig. 3a). There is a significant outward displacement at the intracellular side of TM6, together with a smaller outward movement of TM5 and a rearrangement of TM7 around the NPxxY motif (Fig. 3b, d).
Like the active states of rhodopsin and the β2AR, the active M2 receptor shows rearrangements of the highly conserved DRY motif at the intracellular side of TM3 and the NPxxY motif in TM7 (Fig. 3c, d). In the active state of M2, Arg1213.50 of the DRY motif adopts an extended conformation virtually identical to that seen in metarhodopsin II and the β2AR-Gs complex (Fig. 3c, e, superscript numerals refer to the Ballesteros-Weinstein numbering system), and Asp1203.49 is stabilized by a hydrogen bond with Asn582.39 (Fig. 3c). To assess the importance of Asn582.39 for stabilization of the active conformation, we mutated it to alanine. The resulting mutant displayed normal ligand binding properties, but impaired ability to activate G protein (Extended Data Fig. 3a; Extended Data Table 2). Hence, it is likely that Asn582.39 either directly stabilizes the active conformation, or engages in direct interactions with G protein.
Similar to the DRY motif, the NPxxY region in TM7 shows significant rearrangements on activation (Fig. 3d). Most striking is a partial “unwinding” of TM7 around Tyr4407.53. This positions Tyr4407.53 of the NPxxY motif in close proximity to the highly conserved residue Tyr2065.58 (Fig. 3d). Although these two residues are not close enough to directly interact, their proximity may allow formation of a water-mediated hydrogen bond, as seen in the active-state structures of the β2AR18 and rhodopsin12. Indeed, the position of these two tyrosine residues is highly similar in the active structures of rhodopsin, β2AR, and the M2 receptor (Fig. 3f), suggesting that this feature represents a hallmark of GPCR activation. In addition, a molecular dynamics study recently predicted that Tyr2065.58 and Tyr4407.53 interact in the active conformation of the M2 receptor19, although this model was in other ways dissimilar from the structures presented here.
To assess the importance of this interaction for M2 receptor activation, we mutated Tyr2065.58 to phenylalanine, eliminating its ability to interact with Tyr4407.53 via a bridging water molecule. The Y206F mutant receptor could no longer be activated by acetylcholine (Extended Data Fig. 3a) and gave only a very weak functional response upon treatment with iperoxo. In addition, agonist affinity was reduced by greater than 10-fold (Extended Data Table 2), while antagonist binding was largely unaffected. These results suggest that the Tyr2065.58-Tyr4407.53 interaction stabilizes the active conformation of the receptor in a manner reminiscent of the “ionic lock” interaction20, which stabilizes the inactive conformation of family A GPCRs.
While activation of the β2AR and rhodopsin is associated with modest conformational changes in the orthosteric ligand-binding site, striking structural changes are observed in the M2 receptor. The activated M2 receptor shows a small orthosteric binding site, which completely occludes the agonist iperoxo from solvent (Fig. 4a, b). Indeed, the muscarinic inverse agonist quinuclidinyl benzilate (QNB) is too large to be accommodated in this binding cavity, perhaps accounting for its ability to suppress basal activity of the M2 receptor.
Within the active orthosteric binding pocket, the agonist iperoxo adopts a bent conformation (Fig. 4c; Extended Data Fig. 4). TM helices 5, 6, and 7 move inward, toward the agonist, in the active M2 conformation. TM3, in contrast, undergoes a slight rotation about its axis, but has almost no inward motion toward the ligand. The largest differences between inactive and active states of the M2 receptor involve TM6, where an inward movement of 2 Å at the α-carbon of Asn4046.52 allows for formation of a hydrogen bond between its side chain and iperoxo.
Despite these activation-related structural changes, polar contacts between the agonist iperoxo and the receptor resemble those with QNB bound to the inactive M2 receptor. In particular, the conserved Asp1033.32 serves as a counter-ion to the ligand amine in both cases, and Asn4046.52 engages in hydrogen bonding with both ligands. The smaller size of iperoxo relative to QNB results in more limited hydrophobic contacts, however. This is particularly true along TM5, which engages the phenyl rings of QNB, but makes more limited hydrophobic contact with iperoxo in the active receptor conformation.
The hydrogen bond between Asn4046.52 and the iperoxo isoxazoline oxygen is analogous to the hydrogen bond between this residue and the QNB carbonyl in the inactive receptor state; however, the smaller size of iperoxo necessitates an inward motion of TM6 (Fig. 4d, e). To investigate the role of this hydrogen bond in receptor activation, we mutated Asn4046.52 to glutamine, which, due to the longer side chain, would allow TM6 to form a hydrogen bond with iperoxo in the inactive receptor. Consistent with a previous mutagenesis study21, the N404Q mutant receptor failed to bind detectable amounts of [3H]-NMS, but retained the ability to specifically bind [3H]-QNB, although with 163-fold reduced affinity (Extended Data Table 2). Similarly, the binding affinities for acetylcholine (ACh) and iperoxo were reduced, and while the N404Q mutant was able to activate G protein in response to both iperoxo and ACh, the concentration-response curves were shifted to the right by about 100-fold (Extended Data Fig. 3a), likely due to the reduced agonist binding affinities. Nevertheless, it remains possible that a structural reorientation of Asn4046.52 also contributes to M2 receptor activation.
Like Asn4046.52, Asp1033.32 plays a central role in receptor binding to iperoxo, engaging the trimethyl ammonium ion. Cation-π interactions with Tyr1043.33, Tyr4036.51 and Tyr4267.39 form an aromatic lid over the ligand amine (Fig. 4f). To assess the contribution of Asp1033.32 to receptor activation, we generated and analyzed the D103E mutant M2 receptor, which abolished agonist-induced M2 receptor activation (Extended Data Fig. 3a). The D103E mutant receptor bound [3H]-NMS with WT-like affinity but showed greatly reduced affinities for ACh (~120-fold) and iperoxo (~380-fold) (Extended Data Table 2), indicating that Asp1033.32 recognition of the ligand cation plays a critical role in both agonist binding and receptor activation.
In the active state of the M2 receptor, the inward motion of the upper portion of TM6 allows Tyr4036.51 to hydrogen bond with Tyr1043.33, which in turn hydrogen bonds to Tyr4267.39 (Fig. 4f), resulting in closure of the aforementioned tyrosine lid over the agonist. Hydrogen bonding of this lid appears to be an important feature of agonist binding and activation in muscarinic receptors: mutation of any of the three tyrosines to Phe leads to impaired agonist binding in the homologous M3 muscarinic receptor22, and mutation of Tyr1043.33 and Tyr4036.51 in the M2 receptor has a similar effect23,24. It should be noted that the structure of active M2 receptor bound to other agonists, including acetylcholine, might show differences as compared to the iperoxo-bound structure presented here.
Muscarinic receptors have long served as important model systems for understanding allosteric modulation of GPCR signaling5,6,25. The structures of the inactive M2 and M3 receptors confirmed that these receptors possess a large extracellular vestibule, which has been shown to bind to allosteric modulators26,27. Situated directly above (i.e., extracellular to) the orthosteric site, this cavity also shows a substantial contraction upon activation of the M2 receptor due to the rotation of TM6 (Fig. 4b). The motion of TM6 thus provides a structural link among three regions of the receptor: the extracellular vestibule, the orthosteric binding pocket, and the intracellular surface. The structural coupling of these three regions likely accounts for the fact that allosteric modulators can affect the affinity and efficacy of orthosteric ligands and can also directly activate G proteins as allosteric agonists28.
To better understand how allosteric modulators act at GPCRs, we crystallized the iperoxo-occupied M2 receptor with LY2119620, a positive allosteric modulator (Fig. 5a). This agent has not been studied previously, so we characterized its affinity for the M2 receptor and its allosteric interaction with iperoxo (see Supplementary methods). Radioligand binding assays revealed that LY2119620 has similar pharmacological properties to its congener, LY203329829 (Extended Data Fig. 3b; Extended Data Table 3). It exhibits strong positive cooperativity with iperoxo, and mild negative cooperativity with the inverse agonist [3H]-NMS. While LY2119620 enhances the affinity of the M2 receptor for iperoxo, it does not significantly change the efficacy of this orthosteric agonist (Extended Data Table 3). We also observed that LY2119620 is capable of directly activating the M2 receptor, albeit with low potency and efficacy relative to iperoxo (Extended Data Table 3).
Crystals of the M2 receptor bound to LY2119620 grew under identical conditions to those without the modulator, and the structure revealed unambiguous electron density for LY2119620 in the extracellular vestibule (Extended Data Fig. 5). The modulator is positioned directly above the orthosteric agonist (Fig. 5b), and it engages in extensive interactions with the extracellular vestibule. Specifically, the aromatic rings of the modulator are situated directly between Tyr177ECL2 and Trp4227.35, forming a three-layered aromatic stack. Importantly, a prior mutagenesis study implicated Tyr177ECL2 as a likely contact for the LY2119620 congener, LY2033298, at the M2 muscarinic receptor29. Several polar interactions are also seen (Fig. 5c). In particular, Tyr802.61, Asn4106.58, and Asn419ECL3 form hydrogen bonds to the modulator, and Glu172ECL2 engages in a charge-charge interaction with the ligand piperidine. LY2119620 binds at a site directly superficial to the orthosteric site, separated only by the tyrosine lid, with Tyr4267.39 interacting with both ligands.
The structure of the M2-iperoxo-LY2119620 complex is largely the same as that of receptor and agonist without LY2119620, suggesting that the allosteric binding site is largely pre-formed in the presence of agonist. The extracellular vestibule shows a slight additional contraction around the allosteric ligand (Extended Data Fig. 6). This subtle change stands in contrast to the substantial closure of the extracellular vestibule in the two active structures relative to the inactive conformation (Fig. 5d). A notable exception is Trp4227.35, which adopts a vertical conformation in the presence of LY2119620 and a horizontal conformation with iperoxo alone (Extended Data Fig. 6b). The vertical conformation of this residue in the M2-iperoxo-LY2119620 complex allows it to engage in an aromatic stacking interaction with the modulator, consistent with mutagenesis results implicating Trp4227.35 in the binding of other allosteric modulators30. The effect of mutagenesis of Trp4227.35 on LY2119620 affinity has not been tested, however. Closure of the LY2119620 binding site in the agonist-bound M2 receptor allows far more extensive interactions with the modulator than the inverse agonist-bound conformation (Fig. 5e), likely accounting for the ability of the modulator to enhance agonist binding affinity by preferentially slowing agonist dissociation.
The closed, active conformation of the extracellular vestibule is largely the consequence of the inward motion of TM6, which directly contacts the allosteric modulator, the orthosteric agonist, and likely the G protein as well. Stabilization of the closed extracellular vestibule by LY2119620 and other allosteric modulators may directly stabilize the open, active conformation of the intracellular side of TM6, accounting for the phenomenon of allosteric agonism in addition to positive cooperativity with orthosteric agonists. However, while the differences in TM6 between inactive and active structures can be described as a rigid-body motion, we cannot exclude the possibility that TM6 is flexible, allowing independent conformational changes in the G-protein binding site, the orthosteric site, and the extracellular vestibule.
The structures presented here offer insights into the structural basis for muscarinic receptor activation, and allosteric modulation by a drug-like molecule. In contrast to rhodopsin and the β2AR, extensive changes are seen in the orthosteric binding site and in the extracellular vestibule upon M2 receptor activation. The structure of active M2 receptor bound to the allosteric modulator LY2119620 definitively establishes the extracellular vestibule as an allosteric binding site, and shows that the allosteric modulator induces few additional structural changes as compared to those seen with orthosteric agonist alone. The structures presented here offer only a single view of an active muscarinic receptor; more work will be required to identify additional active states that may exist. Nonetheless, the information presented here provides a structural framework for future studies of GPCR activation and allostery, and may facilitate the development novel therapeutics.
Agonist-induced activation of the human M2 muscarinic receptor was studied in IP accumulation assays as described31. For M2 activation studies, HEK 293 cells were transiently cotransfected with cDNAs encoding the human M2 receptor (Missouri S&T cDNA Resource Center, Rolla, MO) and the hybrid G-protein Gαqi5 (Gαq protein with the last five amino acids at the C-terminus replaced by the corresponding sequence of Gαi; gift from The J. David Gladstone Institutes, San Francisco, CA)32. Twenty-four hours after transfection, cells were transferred into 24 well plates at a density of 100,000 cells per well in a volume of 270 μL. After addition of 30 μL of myo-[3H]inositol (specific activity = 22.5 Ci/mmol, PerkinElmer, Rodgau, Germany), cells were incubated for 15 hrs. Then, medium was aspirated, the cells were washed with serum-free medium supplemented with 10 mM LiCl, and test compounds (diluted in serum-free medium supplemented with 10 mM LiCl) were added at 37 °C for 60 min. Cells were then lysed by adding 150 μL of ice-cold 0.1 M NaOH for 5 min. After neutralisation with 50 μl of 0.2 M formic acid, the cell extract was diluted in buffer (5 mM sodium tetraborate, 0.5 mM Na-EDTA) and separated by anion-exchange chromatography using an AG1-X8 resin (Bio-Rad, Munich, Germany). After washing with water and elution-buffer A (5 mM sodium tetraborate, 60 mM sodium formate) and again with water, total IP was eluted with 2.5 mL elution-buffer B (1.0 M ammonium formate) and directly collected into scintillation counting vials. Radioactivity was measured by scintillation counting after adding 2.5 ml of Emulsifier-Safe (PerkinElmer, Rodgau, Germany). Data were analysed by normalizing dpm values with 0% for the non-stimulated receptor and 100% for the full effect of the reference iperoxo. Dose-response curves were calculated by non-linear regression using the Graphpad Prism 5 software.
Irreversible activation of the M2 receptor was tested at 1 nM FAUC123 in comparison to the reversible ligand iperoxo (1 nM). After incubation for 30 min, the antagonist atropine (1 μM) was added to one half of the sample (buffer was added to the other half) and incubations were continued for an additional 90 min. Total IP accumulation was determined as described above.
To characterize the allosteric interaction between LY2119620 and iperoxo, we performed radioligand binding and cellular functional assays at the wild-type human M2 muscarinic receptor stably expressed in a CHO FlpIn cell line. Increasing concentrations of LY2119620 caused a modest reduction in the specific binding of the orthosteric antagonist, [3H]-NMS, indicating weak negative cooperativity, but robustly enhanced the potency of iperoxo to compete for [3H]-NMS binding, indicating positive cooperativity with the agonist (Extended Data Fig. 3b). Application of an allosteric ternary complex model33,34 to these data yielded the values shown in Extended Data Table 3 for ligand affinity and cooperativities with agonist and antagonist. We then investigated the functional effect of LY2119620 on M2 muscarinic receptor signaling via monitoring receptor-mediated [35S]-GTPγS binding to activated G proteins, or phosphorylation of ERK1/2. [35S]-GTPγS binding was chosen as a proximal measure of receptor activation, while the pERK1/2 assay was chosen because it measures a downstream response that is also a point of convergence of multiple cellular pathways, some of them potentially G protein-independent. In both instances, LY2119620 caused receptor activation in its own right, indicating that the modulator can act as an allosteric agonist, while simultaneously enhancing the potency of iperoxo (Extended Data Fig. 3b). Application of an operational model of allosterism35 to these data yielded the parameter values shown in Extended Data Table 3. Comparison of the binding and functional data indicated that there was no significant difference between any of the pKB estimates of the affinity of LY2119620 for the allosteric site on the free receptor between assays. There was also no significant difference between the cooperativity factors with iperoxo across the assays, indicating that the molecular mechanism of action of LY2119620 is consistent with positive modulation of agonist affinity only, with minimal additional effects on agonist efficacy. This is in contrast to the more complex behavior previously noted with the congener, LY2033298, at the M2 receptor29. Full methods details are available in the Supplementary Methods.
The human M2 muscarinic receptor gene was modified to remove glycosylation sites, and to add an amino-terminal FLAG epitope tag and a carboxy-terminal 8×His tag. In addition, residues 233 – 374 of intracellular loop 3 were deleted. This region has previously been shown to be unstructured36 and is not essential for G protein coupling in the homologous M1 muscarinic receptor37. This construct was expressed in Sf9 insect cells using the BestBac baculovirus system (Expression Systems; Davis, CA). Cells were infected at a density of 4 × 106 cells/mL and then incubated for two days at 27 °C. Receptor was extracted and purified in the manner described previously for the M3 muscarinic receptor8. Briefly, receptor was purified by Ni-NTA chromatography, FLAG affinity chromatography and size exclusion chromatography.
M2 receptor was prepared as described above, and bound to iperoxo by including it at 10 μM starting at FLAG wash steps and in all subsequent buffers. Receptor was reconstituted into phospholipid vesicles composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and Lipid A in a 10:1 (w:w) ratio, then aliquoted at 1 mg/mL receptor concentration and frozen in 100 μL aliquots prior to injection.
M2 receptor was purified as described above with 1 μM atropine included in all buffers. Receptor was then labeled with a 5-fold molar excess of biotin-NHS ester (Sigma-Aldrich; St. Louis, MO) in buffer containing 25 mM HEPES pH 7.2. Following a 30 min incubation at room temperature and a 30 min incubation on ice, unreacted label was quenched with 50 mM tris pH 8. Directly labeled samples with fluorophore-NHS esters were prepared in a similar manner. Receptor was then desalted into buffer containing either 10 μM tiotropium, 10 μM iperoxo, or buffer containing no ligand. Receptor eluted in buffer containing no ligand was treated with 50 μM iperoxo mustard (FAUC123; see Supplementary Information for details) for 20 min at room temperature. Samples were then concentrated, aliquoted, and flash frozen with 20% (v/v) glycerol.
M2 receptor for crystallization was prepared as described above. When bound to FLAG resin, the sample was washed with a mix of dodecyl maltoside buffer (DDM) and buffer containing 0.2% lauryl maltose neopentyl glycol detergent (MNG; Anatrace). These buffers were mixed first in a 1:1 ratio (DDM:MNG buffer), then 1:4, and 1:10 ratios. At each step the 5 mL column was washed with 10 mL of buffer at a 1 mL/min flow rate, and all buffers contained 1 μM atropine. Finally, the column was washed with 10 mL MNG buffer, and then 10 mL of low detergent buffer with agonist (0.01% MNG, 0.001% cholesterol hemisuccinate, 20 mM HEPES pH 7.5, 100 mM NaCl, 10 μM iperoxo). The sample was eluted, mixed with a 1.5-fold stoichiometric excess of Nb9-8 and a second nanobody, NbB4. This nanobody binds to an epitope different from Nb9-8, but was not resolved in the crystal structure. Following mixing, the sample was incubated 30 min on ice, then concentrated and purified by size exclusion in low detergent buffer. Eluted protein was concentrated to A280 = 96, and frozen in liquid nitrogen in 7 μL aliquots.
One Llama (Lama glama) was immunized for six weeks with 1 mg receptor in total. Peripheral blood lymphocytes were isolated from the immunized animal to extract total RNA. cDNA was prepared using 50 μg of total RNA and 2.5 μg of oligo-dN6primer. Nanobody open reading frames were amplified as described38.
Nanobody VHH fragments were amplified by PCR using the primers pYalNB80AMPF (CATTTTCAAT TAAGATGCAG TTACTTCGCT GTTTTTCAAT ATTTTCTGTT ATTGCTAGCG TTTTAGCAAT GGCCCAGGTG CAGCTGCAGG AG) and pYalNB80AMPR (CCACCAGATC CACCACCACC CAAGTCTTCT TCGGAGATAA GCTTTTGTTC GGATCCTGAG GAGACGGTGA CCTGGGTCCC). The PCR products were then co-transformed with linearized pYal into yeast strain EBY100 as for the Nb80 affinity-maturation library, yielding a library size of 0.6×108 transformants.
For the first round of selection, counter-selection was performed against the β2 receptor to remove yeast clones that bind non-specifically to membrane proteins or to secondary staining reagents. 1.0×109 of induced yeast were washed with PBEM buffer and then stained in 5 mL of PBEM buffer containing 1 μM biotinylated β2 receptor liganded with carazolol for 1 hr at 4 °C. Yeast were then stained with streptavidin-647 as a secondary reagent and magnetically-labeled with anti-647 microbeads (Miltenyi) as described previously18. Positively-labeled yeast were then removed by the use of an LD column (Miltenyi); the cleared flow-through was then used for subsequent selection. Positive selection for clones recognizing the active state of the M2 receptor was performed by staining with 2 μM biotinylated M2 receptor bound to the agonist iperoxo in 5 mL PBEM buffer supplemented with 2 μM iperoxo for 1 hr at 4°C. Yeast were then washed, stained with streptavidin-647, and magnetically-labeled with anti-647 microbeads, including 1 μM iperoxo in the PBEM buffer at all steps. Magnetic separation of M2 receptor-binding yeast clones was performed using an LS column (Miltenyi) following the manufacturer's instructions. Magnetically sorted yeast were resuspended in SDCAA medium and cultured at 30°C. Rounds 2-4 were selected in a similar manner, counter-selecting against 1 μM biotinylated β2 receptor bound to carazolol and positively selecting using 1 μM biotinylated M2 receptor bound to iperoxo. For these rounds, the scale was reduced ten-fold to 1 × 108 induced yeast and staining volumes of 0.5 mL.
Conformational selection was performed for rounds 5-9. For rounds 5-8, yeast were stained with 1 μM biotinylated M2 receptor pre-incubated with the high-affinity antagonist tiotropium for 1 hr at 4°C. Yeast were then fluorescently labeled with either streptavidin-647 or streptavidin-PE, and magnetically labeled with the corresponding anti-647 or anti-PE microbeads (Miltenyi). Depletion of inactive-state binders was carried out using an LS column. The cleared yeast were then positively selected by staining with 0.5 μM (rounds 5-7) or 0.1 μM (round 8) M2 receptor pre-bound to iperoxo for 1 hr at 4° C. Yeast were then fluorescently-labeled with either streptavidin-PE or streptavidin-647, using a fluorophore distinct from that used in the previous counter-selection step. Magnetic separation of agonist-occupied M2 receptor was performed using an LS column, as for steps 1-4. For round 9, two-color FACS was performed. Induced yeast were simultaneously stained with 1 μM Alexa647-labeled M2 receptor reacted with iperoxo mustard and 1 μM Alexa488-labeled M2 receptor pre-bound with tiotropium for 1 hr at 4°C. Alexa647 positive/Alexa488 negative yeast were purified using a FACS Jazz cell (BD Biosciences) sorter. Post-sorted yeast were plated onto SDCAA-agar plates and the nanobody-encoding sequences of several colonies were sequenced. Full sequences of clones confirmed to enhance agonist affinity are below:
|Clone||Full amino acid sequence|
|Nb9-1||QVQLQESGGG LVQAGGSLRL SCAASGHTFS SARMYWVRQA PGKEREFVAA ISRSGFTYSA DSVKGRFTIS RDIANNTVYL QMNSLQPEDT AIYTCYAAYL DEFYNDYTHY WGLGTQVTVS S|
|Nb9-8||QVQLQESGGG LVQAGDSLRL SCAASGFDFD NFDDYAIGWF RQAPGQEREG VSCIDPSDGS TIYADSAKGR FTISSDNAEN TVYLQMNSLK PEDTAVYVCS AWTLFHSDEY WGQGTQVTVS S|
|Nb9-20||QVQLQESGGG LVQPEGSLTL ACDTSGFTMN YYAIAWFRQA PEKEREGLAT ISSIDGRTYY ADSVKGRFTI SRDSAKNMVY LQMNNLRPED TAVYYCSAGP DYSDYGDESE YWGQGTQVTV SS|
Nanobody sequences were subcloned into a modified pMalp2x vector (New England Biolabs), containing an amino-terminal, 3C protease-cleavable maltose binding protein (MBP) tag and a carboxy-terminal 8×His tag. Plasmids were transformed into BL21(DE3) cells and protein expression induced in Terrific Broth by addition of IPTG to 1 mM at an OD600 of 0.8. After 24 hr of incubation at 22°C, cells were harvested and periplasmic protein was obtained by osmotic shock. MBP-nanobody fusions were purified by Ni-NTA chromatography and MBP was removed using 3C protease. Cleaved MBP was separated from the 8×His tagged nanobodies by an additional Ni-NTA purification step. The 8×His tag was subsequently removed using carboxypeptidase A.
Heterotrimeric Gi was prepared by expression using a single baculovirus for the human Gαi1 subunit and a second, bicistronic virus for human Gβ1 and Gγ2 subunits. G protein was expressed in HighFive insect cells, and then purified as described previously for Gs10. In brief, G protein was extracted with cholate, purified by Ni-NTA chromatography, detergent exchanged into dodecyl maltoside buffer, and then purified by ion exchange and dialyzed prior to use.
M2 receptor was expressed and purified as described above. Receptor was then reconstituted into HDL particles consisting of apolipoprotein A1 and a 3:2 (mol:mol) mixture of the lipids POPC:POPG (1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1′-rac-glycerol) respectively, Avanti Polar Lipids). Binding reactions contained 50 fmol functional receptor, 0.6 nM [3H] N-methyl scopolamine (NMS), 100 mM NaCl, 20 mM HEPES pH 7.5, 0.1% BSA, and ligands and nanobodies as indicated. Concentration-dependent effects of nanobodies were measured in the presence of 10 nM iperoxo. All reactions were carried out in a 500 μL volume. For samples containing G protein, purified Gi heterotrimer from insect cells was added to the reactions at a 1000-fold dilution from a 200 μM stock, resulting in a large stoichiometric excess over receptor and diluting G protein below the detergent CMC to allow incorporation into HDL particles, essentially as described previously39. Reactions were mixed and then incubated for 2 hr. Samples were then filtered on a 48-well harvester (Brandel) onto a filter which had been pre-treated with 0.1% polyethylenimine. All measurements were taken by liquid scintillation counting, and experiments were performed at least in triplicate.
A mammalian expression plasmid coding for the human M2 muscarinic receptor (M2R-pcDNA3.1+) was obtained from the Missouri S&T cDNA Resource Center. Mutant M2 receptors were generated by using the QuikChange site-directed mutagenesis kit (Stratagene, Foster City, CA, USA) according to the manufacturer's instructions. The identity of all mutant M2 receptor constructs was confirmed by DNA sequencing.
WT and mutant M2 receptors were transiently expressed in COS-7 cells grown in 100 mm dishes, as described previously40. For functional studies, the various receptor constructs (3 μg each) were co-expressed with a chimeric G protein α subunit (Gqi5; 3 μg plasmid DNA) in which the last five amino acids of Gαq were replaced with the corresponding Gαi sequence41.
Acetylcholine (ACh) bromide was purchased from Sigma (St. Louis, MO, USA). Iperoxo was a generous gift of Bristol Myers Squibb (New York, NY). [3H]-NMS (85.5 Ci/mmol) and 3-quinuclidinyl benzilate ([3H]-QNB; 47.4 Ci/mmol) were from PerkinElmer Life Sciences (Downers Grove, IL, USA). Radioligand binding studies were carried out with membranes prepared from transfected COS-7 cells as described41. Forty-eight hours after transfection, cells were harvested and resuspended in 25 mM sodium phosphate buffer (pH 7.4) containing 5 mM MgCl2. Membrane homogenates were prepared and resuspended in the same buffer. [3H]-NMS or [3H]-QNB binding reactions were carried out in the presence of 9 μg of membrane protein for 3 hr at room temperature (total volume of the incubation mixture: 0.5 ml). In saturation binding studies, six different concentrations of the radioligand were used ([3H]-NMS, 0.3 nM to 10 nM; [3H]-QNB, 0.05 nM to 20 nM). In competition binding assays, membrane homogenates were incubated with ten different concentrations of ACh (13 nM to 1 mM) or iperoxo (0.13 nM to 10 μM) in the presence of a fixed concentration of radioligand (2 nM [3H]-NMS for all receptors except N404Q; 15 nM [3H]-QNB for N404Q and 0.5 nM [3H]-QNB for WT M2 receptor). Non-specific binding was determined in the presence of 10 μM atropine. Reactions were stopped by rapid filtration through GF/C filters. Data were analyzed using Prism 4.0 software (GraphPad Software, Inc., San Diego, CA).
COS-7 cells co-expressing WT or mutant M2 receptor and the hybrid G protein, Gqi541, were incubated with increasing concentrations of agonists (ACh, 5 nM to 50 μM; iperoxo, 50 pM to 0.5 μM), and increases in intracellular calcium levels were determined in 96-well plates using FLIPR technology (Molecular Devices, Sunnyvale, CA), as described in detail previously42,43. Agonist concentration-response curves were analyzed using Prism 4.0 software.
Purified M2 receptor was reconstituted into lipidic cubic phase by mixing with a 1.5-fold excess by mass of 10:1 (w:w) monoolein cholesterol lipid mix. Protein and lipid were loaded into glass syringes (Art Robbins Instruments, Sunnyvale, CA), and then mixed 100 times by the coupled syringe method44. Samples of 30 – 100 nL in volume were spotted onto 96 well glass plates and overlaid en bloc with 600 nL precipitant solution for each well. Precipitant solution consisted of 10 – 20% PEG300, 100 mM HEPES pH 7.2 – 7.9, 1.2% 1,2,3-heptanetriol, and 20 – 80 mM EDTA pH 8.0. Identical conditions were used to crystallize LY2119620-receptor complexes, except that the overlay precipitant solution was supplemented with 500 μM LY2119620. Crystals grew in 24 hr, and reached full size within two days. Crystals were then harvested in mesh grid loops (MiTeGen, Ithaca, NY) with 10 – 50 crystals per loop and stored in liquid nitrogen prior to use.
Grids of crystals were rastered at Advanced Photon Source beamlines 23ID-B and 23ID-D. Initial rastering was performed with an 80 μm by 30 μm beam with 5-fold attenuation and 1 sec exposure, and regions with strong diffraction were sub-rastered with a 10 μm collimated beam with equivalent X-ray dose. Data collection was similarly performed with a 10 μm beam, but with no attenuation and exposures of typically 1 – 5 s. An oscillation width of 1 – 2 degrees was used in each case, and wedges of 5 – 10 degrees were compiled to create the final data sets.
Diffraction data were processed in HKL200045, and statistics are summarized in Extended Data Table 1. The structure was solved using molecular replacement with the structure of the inactive M2 receptor (PDB ID: 3UON) and Nb80 (PDB ID: 3P0G) as search models in Phaser46. The resulting structure was iteratively refined in Phenix47 and manually rebuilt in Coot48. Final refinement statistics are summarized in Extended Data Table 1. Figures were prepared in PyMol (Schrödinger).
We acknowledge support from the National Science Foundation (graduate fellowship to A.C.K., and Award 1223785 to B.K.K) , the Stanford Medical Scientist Training Program (A.M. and A.M.R.), the American Heart Association (A.M.), the Ruth L. Kirschstein National Research Service Award (A.M.R.), National Institutes of Health grants NS02847123 and GM08311806 (B.K.K.), the Mathers Foundation (B.K.K., W.I.W., and K.C.G), the Deutsche Forschungsgemeinschaft for the grant GM 13/10-1 (K.E., H.H., P.G.), the National Health and Medical Research Council (NHMRC) of Australia program grant 519461 (P.M.S. and A.C.), NHMRC Principal Research Fellowships (P.M.S. and A.C.), and the Howard Hughes Medical Institute (K.C.G.). This work was supported in part by the Intramural Research Program, NIDDK, NIH, US Department of Health and Human Services (J.H., K.H., and J.W.). We thank Katie Leach for performing ERK assays, Briana Davie and Peter Scammells for synthesis of iperoxo. We thank Hongling Xiao, Carrie H. Croy, Douglas A. Schober for functional characterization of LY2119620. We thank Tong Sun Kobilka for preparation of affinity chromatography reagents and Foon Sun Thian for help with cell culture.
Full Methods and associated references are available in the online version of the paper at www.nature.com/nature.
Author Contributions: A.C.K. expressed and purified M2 receptor for yeast display and crystallographic experiments, performed crystallization, data collection, and structure refinement, and performed radioligand binding assays to validate nanobody activity. A.C.K. A.M.R. and A.M. designed experiments to identify nanobodies by yeast display. A.M.R. performed all yeast selections, and expressed and purified Nb9-8 and other nanobodies. J.H. and K.H. performed site-directed mutagenesis and characterization of resulting mutants. K.E. synthesized FAUC123. H.H. performed cell assays and radioligand binding to characterize FAUC123. C.V. performed pharmacological characterization of LY2119620. P.M.S. and A.C. supervised pharmacological characterization of LY2119620. C.C.F. designed key solubility, physical chemistry and ligand analysis to select LY2119620 as an appropriate co-crystallization candidate for the M2 receptor. P.G. supervised synthesis and characterization of FAUC123. E.P. and J.S. performed llama immunization, cDNA production, and performed selections by phage display. W.I.W. supervised structure refinement. K.C.G. supervised yeast selection experiments. J.W. supervised mutagenesis experiments and analyzed results. B.K.K. provided overall project supervision, and with A.C.K., A.M.R., and A.M. wrote the manuscript with assistance from A.C. and J.W.
Author Information: Coordinates and structure factors for the active M2 receptor in complex with Nb9-8 and iperoxo are deposited in the Protein Data Bank under accession code 4MQS, and the coordinates and structure factors of the same complex bound additionally to the allosteric modulator LY2119620 are deposited under accession code 4MQT.