Μor (µ-opioid receptor) –GI complex structure _nature2018_antoine Koehl, Hongli Hu, Shoji Maeda, etc.

#原文标题, authors, periodicalsStructure of theμ-opioid receptor–gi protein complex

• Antoine Koehl,
• Hongli Hu,
• Shoji Maeda,
• Yan Zhang,
• Qianhui Qu,
• Joseph M. Paggi,
• Naomi R. Latorraca,
• Daniel Hilger,
• Roger Dawson,
• Hugues Matile,
• Gebhard F. X. Schertler,
• Sebastien Granier,
• William I. Weis,
• Ron O. Dror,
• Aashish Manglik,
• Georgios Skiniotis
• Brian K. Kobilka.

Nature Volume 558, pages 547–552 (2018)

#总结

#本文前置: Structural insights intoμ-opioid receptor activation. Nature volume 524, pages 315–321 (August)

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This article contains

Abstract-1

Conclusion-5

Method-9

Figure-5

Extended Data Graph-8, table-2

Structure of Μor (μ-opioid receptor) –gi complex

Summary

Gi:adenylyl cyclase-inhibiting heterotrimeric G-protein adenosine-inhibitor- derived dimer g protein

damgo: [ D -ala2, N -mephe4, Gly-ol]-enkephalin h-tyr- D -ala-gly- N -mephe-gly-oh n→c last glycine carbonyl replaced by hydroxyl group

1. The structure of Μor-damgo and Μor-gi (non-nucleic acid) in the frozen electron microscope, 3.5 horizontal is obtained.

2, Damgo occupies the morphine ligand pocket, n-terminal and the receptor of conservative residues, c-side participation in the selection of opioid ligand important areas.

3, the Μor-gi complex with the previously determined structure of the Gpcrs-gs comparison, got two differences:

(1) The site of the TM6 Helix (the structural changes caused by the substitution of amino acids in the latter middle);

(2) The interaction of G protein α subunit with receptor core.

3.5? Cryo-em Map of Aμor–gi complex

Apyrase: three monophosphate adenosine triphosphate,EC 3.6.1.5 ,NTP---NDP + Pi-, NMP + 2Pi

Scfv:single-chain antibody fragment or Single-chain variable fragment

Gtpγs : gtpgammas (Gtpγs, guanosine 5 '-o-[gamma-thio]triphosphate)

1. Damgo is a synthetic analogue of μor selective stimulant endorphins, and the damgo-μor and Gi1 heterogeneous dimer are incubated together, resulting in apyrase removal of GDP from the compound.

2, Gi even have scfv16,scfv16-gi and damgo-μor through scFv16 stability, scFv16 can also inhibit the gtpγs-mediated separation of nucleic acid-free complexes.

3, using single-particle refrigeration electron microscope to obtain the μor–damgo–gi–scfv16 three-dimensional figure, the nominal resolution of 3.6?

4. ScFv16 combines a composite plane containing αn helix of gαi and Theβ-propeller of Gβ, with a μor–gαi interface distance of more than 20, and no interface interfering with Gα and gβ subunits. (Extended Data Fig. 1)

5, the SCFV16 signal difference will increase the image resolution to 3.5?

6, 3.5? Under the conditions of Μor-damgo,μor-gi. (Fig. 1a, B)

Activation Ofμor by a peptide agonist

Bu72:small molecule Morphinan agonist, combined with complex ring systems

Nb39:active-state stabilizing nanobody in the presence of 5μm Nanobody (Nb39), the affinity of the potent Morphinan Agonist BU72 increases from 470 pm to PM. BU72 has a dissociation half-life of $ min in the presence of Nb39. Nb39 also enhances the affinity ofμor agonists damg O and endomorphin-2, indicating that effect are not limited to morphinan agonists.

1. Previous work has been 2.2? ΜOR-BU72 crystal structure of activated State, NB39 crystal structure.

2, μor-bu72 or μor-Damgo activation State ligand-binding pocket structure and amino acid localization height similar →μor in an unchanging way to identify different structures of stimulants. (Fig. 1c)

3, compared with BU72, the N-end of Damgo occupy a similar joint pocket point, but the C-end and ECL distance far to??。 (Fig. 1d, E)

4, molecular dynamics simulation, in the 1?μs Damgo basically remain unchanged. (Fig. 1f, Extended Data Fig 5) (Figure note, Damgo frequently return to original sites)

5, Damgo N-end and D1473.32 form a solid salt bridge, the same amino also often with the Y3267.43 form hydrogen bonds. (Fig. 1e)

6,μor-bu72 crystal structure shows that Damgo phenol-based and H2976.52 with two water molecules into a bridge, but the simulation shows that one of the water molecules quickly separated, stable ligand combination requires a single water molecule. This is a sign of μ-/δ-/κ-or to identify opioid types. (Extended Data Fig. 6)

7, Damgo combined μor 500 times times stronger than the other two species, selectivity in μ-/δ-or mainly depends on ECL1 residues, κ-or mainly ECL3.

8, Damgo C-end graph density is slightly weaker than N-terminal, and the simulation results corresponding. (Fig. 1f, Extended Data Fig 5)

9, Damgo N(Me) Phe side chain occupy a ECL1 near the conservative hydrophobic pocket, glycine hydroxyl fold to the ligand, this model and μor with the ring endorphins highly affinity. (Fig. 1e)

Structure of Gi-stabilized Activeμor

1, relative to the non-activated state,Μor-gi and Nb39 fixed μor-bu72 has a similar TM6 of the outward displacement (root mean square deviation 1?). (Fig. 2a, b)

2. A large number of highly conserved residues in most GPCR show the importance of activating the receptor, including d3.49r3.50y3.51, n7.49p7.50xxy7.53, and three cores (I3.40, P5.50, F6.44). These structures are almost identical to the nb39-μor complexes in Μor-gi. (Fig. 2c)

3, Nb39 and Gi promote a similar increase in doping affinity, providing a cellular g protein coupling domain and ligand-binding pocket between the transformation of communication.

< Span style= "Font-family:arial, Helvetica, Sans-serif" >< Span style= "Font-family:arial, Helvetica, Sans-serif" > 4, two Nb39 and Gi binding activation status μor are particularly noteworthy:

< Span style= "Font-family:arial, Helvetica, Sans-serif" >< Span style= "Font-family:arial, Helvetica, Sans-serif" > (1) activation of binding with nano-antibodies μor compared to  μor -gi TM6 in complex Displacement  3??。 (fig. 2b)

< Span style= "Font-family:arial, Helvetica, Sans-serif" >< Span style= "Font-family:arial, Helvetica, Sans-serif" >< The structure of span style= "Font-family:arial, Helvetica, Sans-serif" > (2) ICL3 is different. The special ICL3 structure of the μor combined with Nb39 reflects the unique interaction between the nano-antibody and μor, and the difference in the similar ICL3 structure exists in Nb80 β2-adrenergic (receptor), which Β2ar and Gs interoperate. (fig. 2b)

< Span style= "Font-family:arial, Helvetica, Sans-serif" >< Span style= "Font-family:arial, Helvetica, Sans-serif" >< Span style= "Font-family:arial, Helvetica, Sans-serif" > 5, in the state of binding with G protein, < Span style= "Font-family:arial, Helvetica, Sans-serif" > Β2ar TM6 9??。 outward than μor (fig. 2d)

Structural changes in Gi

Structural insights into gi-coupling specificity of theμor

Method

Purification Ofμor

Expression and purification of heterotrimeric Gi

Generation of ScFv16

Formation and purification of theμor-gi-scfv16 complex

Cryo-em and 3D reconstructions Ofμor-gi-scfv16 complex

Model Building and refinement

System Setup for Molecular dynamics simulations

Molecular Dynamics simulation Protocols

Analysis protocols for molecular dynamics simulation

Μor (µ-opioid receptor) –GI complex structure _nature2018_antoine Koehl, Hongli Hu, Shoji Maeda, etc.