Crystal structure of the μ-opioid receptor bound to a morphinan antagonist

Summary Opium is one of the world’s oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many of their undesirable side effects (sedation, apnea and dependence) by binding to and activating the G-protein-coupled μ-opioid receptor (μOR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the μOR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most GPCRs published to date, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the μOR crystallizes as a two-fold symmetric dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.


Supplementary Figure 3. Electron density within the ligand-binding pocket.
The ligand β-FNA is bound within a µOR ligand-binding pocket and covalently tethered to K233 5.39 . a. Maps for binding pocket residues and for two water molecules are shown with mesh depicting the 2F o -F c electron density contoured at 1.3 σ. b. The same view of the binding pocket is shown with an omit map of β-FNA and the K233 5.39 side chain atoms contoured at 3.0 σ. The electron density supports the previously characterized covalent link between the side chain amine of K233 5.39 and the highly electrophilic carbon of β-FNA 48 . c. Two more views of the electron density around the β-FNA ligand with the covalently tethered K233 5.39 side chain are shown. The density provides sufficient features to reliably position the cyclopropyl moiety and the core morphinan group. Superscripts indicate Ballesteros-Weinstein numbers.

Supplementary Figure 4. Intracellular domain of µOR.
Although the highly conserved residues of the DRY motif in the µOR make similar contacts to those seen in the β 2 AR, the relative positions of TM3, TM5, and TM6 are similar to rhodopsin. The intracellular side of µOR more closely resembles rhodopsin (a) as compared to β 2 AR (b). A closer view of the region around the DRY motif is shown for each receptor in c,d,e. In rhodopsin, the highly conserved R135 3.50 residue forms an ionic interaction with D134 3.49 and E247 6.30 c. In both the µOR and β 2 AR, the conserved aspartate and arginine of the DRY motif recruit a polar residue from ICL2 to form an extended bonding network. However, in the µOR, T279 6.34 engages in a polar interaction with R165 3.50 .

Supplementary Figure 5. Residues critical for DAMGO binding.
The high-affinity, µOR selective peptide DAMGO binds in a pocket that overlaps with β-FNA. Mutation of the residues shown in red severely affects DAMGO binding to µOR. While H297 makes contacts with β-FNA, residues K303, W318, and H319 do not. The binding site for peptides like DAMGO therefore overlaps with morphinan ligands but likely extends further towards the extracellular side of the receptor. Figure 6. Interface TM1-TM2-H8 between two µOR protomers. Although the larger contact between µOR molecules is observed through the TM5-TM6 interface, a more limited interaction is observed with another crystallographically related µOR molecule involving TMs 1-2 and Helix 8. This interface is described in more detail here. a, Side view (top) and extracellular view (bottom) of the TM1-TM2-H8 interface. b, The interface is expanded and shown in detail with interacting residues within 4.2 Å shown as sticks. c, Tomographic representation along the interface viewed from the extracellular side (as indicated in panel c).

Supplementary Figure 7. Interface between µOR-T4L and CXCR4-T4L pairs.
The interface between two µOR-T4L molecules is shown, with the interface for one subunit depicted in cyan and the other in magenta. Each µOR monomer buries 1492 Å 2 of its partnered monomer, resulting in a total buried surface area of 2984 Å 2 for one µOR-µOR pair. The T4L-T4L contacts within the interface are more limited, with each T4L monomer burying 114 Å 2 of its paired T4L. This is in comparison to a smaller buried surface area for the similar interface observed in the CXCR4-T4L crystal structures. In the structure of CXCR4-T4L depicted here (PDB ID: 3ODU), each receptor buries 784 Å 2 of its partnered monomer while the T4L-T4L contact amounts to 287 Å 2 of buried surface area. Within the available CXCR4 structures, the highest buried surface area across this interface is 1077 Å 2 (PDB ID: 3OE0). Buried surface area calculations were performed using the PDBePISA server.

Supplementary Figure 8. Network of interactions for the TM5-TM6 interface and comparison with CXCR4. a.
The four helix bundle interface between TM5-TM6 of two µOR protomers (blue and red). The 28 contacting residues (defined as less than 4.2 Å from each protomer) are shown as sticks. The right panel displays a 90º rotated view of one protomer viewed from the interface. b, The dimeric interface observed in the crystal structure of CXCR4 resembles the µOR interface but with a less extensive network of interacting residues. We show 3OE0 here because it has the most extensive interaction network within available CXCR-T4L structures. The CXCR4 dimeric interface is similar to the one observed for the µOR in that the dimeric interactions towards the extracellular side of the receptor are driven by residues in TM5 and TM6. Unlike µOR, the dimeric interface for CXCR4 may also be driven by residues in TM3 and TM4. However, this full complement of interactions is not observed in all of the available CXCR4 structures, perhaps as a result of different arrangements of T4L at the intracellular side of the receptor. Supplementary Fig. 9: Comparison of the TM5-6 interface between µOR-µOR and µOR-δOR.
a, Alignment of TM5 and TM6 sequences from the µOR and δOR. Interacting residues are shown in bold font, and non-strictly conserved residues are highlighted in yellow. b, Potential µOR-δOR four-helix bundle interface shown in detail with TM5-TM6 of µOR in blue and TM5-TM6 of δOR in red. Yellow sticks indicate residues that are different in δOR as compared to µOR. Here, a homology model of δOR based on the µOR was aligned with the red protomer. c, Tomographic representation along the interface viewed from the extracellular side (as indicated in panel b). The differences between µOR and δOR results in a slightly different dimeric interface but these changes do not result in severe clashes or considerably unfavorable interactions. Therefore, the differences in the residues between µOR and δOR at this interface are likely not sufficient to prohibit an interaction at the TM5-TM6 interface. Figure 10: Comparison of the TM1-TM2-H8 dimer interface between µOR-µOR and µOR-δOR.

Supplementary
While the primary intermolecular contact seen in the crystal structure of µOR occurs along TM5-TM6, an additional parallel contact is seen involving TMs 1-2 and Helix 8. a, Alignment of TM1, TM2 and Helix 8 sequences from the µOR and δOR. Interacting residues are shown in bold font, and non-strictly conserved residues are highlighted in yellow. b, Potential µOR-δOR four-helix bundle interface shown in detail with TM5-TM6 of µOR in blue and TM5-TM6 of δOR in beige. Yellow sticks indicate residues that are different in δOR as compared to µOR. Here, a homology model of δOR based on the µOR was aligned with the beige protomer. c, Tomographic representation along the dimer interface viewed from the extracellular side (as indicated in panel b). As for the TM5-TM6 interface, the differences between µOR and δOR result in a slightly different interaction network predicted for the TM1-TM2-H8 interface shown here.