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Activation and Allosteric Modulation of a Muscarinic Acetylcholine Receptor


Activation and Allosteric Modulation of a Muscarinic Acetylcholine Receptor

Andrew C Kruse et al. Nature.


Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, 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 bound to 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 the activation mechanism and allosteric modulation of muscarinic receptors.


Figure 1
Figure 1. Isolation of Nb9-8
a, Nanobodies from a llama immunized with M2 receptor were displayed on yeast as an amino terminal fusion to Aga2p, and subjected to magnetic selection to enrich clones that bind preferentially to agonist-occupied receptor. b, For selections, an aziridinium ion derivative of iperoxo called FAUC123 was synthesized, allowing covalent modification of the receptor. c, Yeast were stained simultaneously with agonist-occupied M2 receptor and antagonist-occupied receptor labeled with distinct fluorophores. d, Yeast from each selection round (Rd. 1 – 9) were stained in this manner to assess selection progress, showing a clear enrichment first for non-selective binders (upper right quadrants) followed by specific enrichment for agonist-preferring clones (lower right quadrants). *Indicates a selection round employing conformational selection. **Indicates a selection round using FACS.
Figure 2
Figure 2. M2 active-state specific nanobodies
a, Three nanobodies were selected for detailed characterization, each with entirely unique complementarity determining region (CDR) sequences. These three nanobodies were expressed on the surface of yeast, and characterized by flow cytometry staining with FAUC123-bound (i.e., agonist-bound) M2 receptor and tiotropium-bound (i.e., antagonist-occupied) receptor. b, Each of the three clones displayed a preference for agonist-occupied receptor to varying degrees. c, Purified nanobodies were tested in a dose-response assay for their ability to suppress [3H]-NMS binding to the M2 receptor in the presence of 10 nM (IC20) iperoxo, with Nb9-8 being the most potent clone. d, Like the G protein Gi, Nb9-8 caused a substantial enhancement of iperoxo affinity in a competition binding assay.
Figure 3
Figure 3. Intracellular changes on activation of the M2 receptor
a, The overall structure of the active-state M2 receptor (orange) in complex with the orthosteric agonist iperoxo and the active-state stabilizing nanobody Nb9-8 is shown. b, Compared to the inactive structure of the M2 receptor (blue), transmembrane helix 6 (TM6) is substantially displaced outward, and TM7 has moved inward. Together, these motions lead to the formation of the G protein-binding site. c-d, Conserved motifs likewise show substantial changes on activation, and adopt conformations similar to those seen in the two other active GPCR structures (e-f). In particular, an interaction between two conserved tyrosines (Tyr5.58 and Tyr7.53) is likely mediated by a water molecule (blue circle), as seen in the high-resolution structure of the active β2AR [Ref. 18].
Figure 4
Figure 4. Orthosteric ligand binding site
a, Orthosteric ligands used for crystallization of inactive and active M2 receptor are shown in. b, Cross-sections through the receptor are shown, with the interior in black. In the inactive conformation, the receptor (blue, at left) partially encloses the antagonist QNB, while the active conformation receptor encloses the agonist entirely, such that it is completely buried within the receptor (orange, at right). c, Conformational changes within the ligand binding pocket are shown from the extracellular side in, with changes highlighted as red arrows. d, A side view shows the inward motion of TM6, which is required for the formation of a hydrogen bond between Asn4046.52 and the agonist iperoxo. e, Activation thus involves a pivot of TM6, which moves inward in the orthosteric site and outward at the intracellular side. f, The closure of the binding pocket allows the formation of a hydrogen bonded tyrosine lid, located superficial to the agonist.
Figure 5
Figure 5. Structure of a GPCR allosteric modulator complex
a, The M2 receptor occupied by the orthosteric agonist iperoxo was crystallized in complex with the positive allosteric modulator LY2119620. b, The allosteric ligand binds to the extracellular vestibule just above the orthosteric agonist. A cross-section through the membrane plane shows the relative positions of the two ligands. c, Several polar contacts are involved in LY2119620 binding, in addition to extensive aromatic stacking interactions with Trp4227.35 and Tyr177ECL2. d, Upon activation, the M2 receptor undergoes substantial conformational changes in the extracellular surface, leading to a contraction of the extracellular vestibule. e, This creates a binding site that fits tightly around the allosteric modulator, which would otherwise be unable to interact extensively with the extracellular vestibule in the inactive receptor conformation.
Extended Data Figure 1
Extended Data Figure 1. Characterization of FAUC123
a, Activation of M2 receptor by the prototypical muscarinic agonist carbachol, the high affinity agonist iperoxo, and an irreversible iperoxo analog (FAUC 123) shows that iperoxo and FAUC 123 are exceptionally potent full agonists at the M2 muscarinic receptor. Points indicate mean ± SEM of three independent measurements, each performed in triplicate. b, Sf9 membranes expressing the human M2 receptor were incubated overnight at 4 °C with either no ligand, 100 μM iperoxo, or 100 μM FAUC 123. Membranes were then washed three times in buffer without ligand, and incubated with a saturating concentration (20 nM) of [3H]-NMS. Incubation with iperoxo had no effect on radioligand binding, while FAUC 123 blocked almost all [3H]-NMS binding sites. Bars indicate mean ± SEM of three independent measurements. c, FAUC 123 was tested for its ability to induce M2 receptor activation following covalent modification. While iperoxo-induced inositol phosphate production was blocked by 1 μM atropine, FAUC 123-induced activation was not susceptible to atropine blockade. Bars indicate mean ± SEM of three independent measurements.
Extended Data Figure 2
Extended Data Figure 2. Comparison to other active GPCR structures
Structures of all activated GPCRs show similarities in conformational changes at the intracellular surface. In each case, the intracellular tip of transmembrane helix 6 (TM6) moves outward on activation, as seen in the view from the intracellular side (right panels). This creates a cavity to which a G protein can bind the receptor.
Extended Data Figure 3
Extended Data Figure 3. Pharmacology
a, Functional properties are shown for M2 receptors in which key residues were mutated. Agonist-induced increases in intracellular calcium levels were monitored via FLIPR using transfected COS-7 cells. Since some mutant receptors (N58A, D103E) were expressed at lower levels than the WT receptor, reference curves were obtained using cells transfected with either 3 μg DNA or 1 μg WT receptor DNA. The latter cells showed receptor expression levels comparable to those found with the N58A and D103E mutants (see Extended Data Table 2 for details). Data are given as means ± SEM of three independent experiments, each carried out in triplicate. AU, arbitrary units. b, The interaction between LY2119620 and iperoxo was measured by radioligand binding and functional assays. LY2119620 enhances the affinity of iperoxo (top graph) and its signaling potency (bottom graphs), and is also able to directly activate M2 receptor signaling as measured by [35S]GTPγS and ERK1/2 phosphorylation. Experiments were carried out with CHO cells stably expressing the human M2 receptor, and points are shown as mean ± SEM of three independent experiments, each carried out in duplicate.
Extended Data Figure 4
Extended Data Figure 4. Binding site diagram
M2 receptor residues interacting with the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620 are shown. Polar contacts are highlighted as red dotted lines, and hydrophobic contacts are in green solid lines.
Extended Data Figure 5
Extended Data Figure 5. Electron density
a-b, Fo-Fc omit maps are shown in gray, contoured at 2.5 σ within a 2.5 Å radius of the indicated ligand. c-f, 2Fo-Fc maps are shown in blue, contoured at 1.5 σ within a 2.0 Å radius of the indicated region.
Extended Data Figure 6
Extended Data Figure 6. Comparison of M2 receptor structures with and without LY2119620 bound
Comparison of the structure of active M2 receptor with and without the allosteric modulator LY2119620 reveals that there are few differences outside the extracellular vestibule. Within the extracellular vestibule, there is a slight contraction in the presence of the modulator, and Trp4227.35 undergoes a change of rotamer (panel b, red arrow).

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