Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor

Abstract

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

Keywords: GPCR; active state; addiction; crystallography; morphinan; nanobody; opioid receptor; structure-function.

Figure 1. Identification of inactive and active state stabilizing nanobodies and overall structure of the KOP-MP1104-Nb39 complex

(A) Cartoon of receptor-nanobody (Nb) interaction monitored by bioluminescence resonance energy transfer (BRET). Agonist stimulated GPCR is bound by active state stabilizing Nb causing BRET signal. Nanobodies are tagged with C-terminal YFP and GPCRs are tagged with C-terminal Rluc (Renilla luciferase). (B) BRET measurement of SalA-mediated Nb recruitment at KOP. The clones Nb6 and Nb7 (EC₅₀ = 341±15 nM), which have the same protein sequence, show dissociation from the receptor upon SalA stimulation, indicating that Nb6/7 is pre-bound to KOP. The BRET signal from the KOP-Nb39 complex (EC₅₀ = 11.3±1.1 nM) increases with increasing SalA concentrations, indicating that Nb39 recognizes active state KOP (N = 3). (C) (top) Scheme of extended ternary complex model of GPCR activation. R, receptor; L, ligand; R*, active state receptor; X, transducer or transducer mimetics. (middle and bottom) Measurement of saturation binding at KOP with or without Nb39. High-affinity binding sites increase about 6-fold for the full agonist ³H-U69,593 in the presence of Nb39 (Kd = 2.80±0.06 nM, B_max = 5262±138 fmol/mg) compared to KOP alone (Kd = 5.41±0.05 nM, B_max = 860±35 fmol/mg) (N = 3). High-affinity binding sites increase by ~10% for the partial agonist ³H-Diprenorphine in the presence of Nb39 (Kd = 0.26±0.02 nM, B_max = 10315±195 fmol/mg) compared to KOP alone (Kd = 0.84±0.05 nM, B_max = 9191±120 fmol/mg) (N = 3). (D) Ligand-mediated Nb39 binding to KOP measured BRET. EC₅₀s for Nb39 recruitment: MP1104, 0.12±0.02 nM; U69,593, 3.78±0.04 nM; SalA, 11.2±0.8 nM (N = 3). (E) Overall x-ray crystal structure of the KOP-MP1104-Nb39 complex. See also Figure S1.

Figure 2. Large-scale structural changes between inactive and active KOP

(A) Structural alignment of active (blue) and inactive (PDB ID: 4DJH, grey) KOP shows TM6 outward displacement of ~10 Å. (B) Extracellular view highlights contraction of the TMs and extracellular loops upon binding to MP1104 (orange) versus JDTic (purple). Distances were measured between Cα atoms of I58^1.31, Q115^2.60, Q213^ECL2, D223^5.35, L299^6.60 and S310^7.33. (C) Intracellular view shows expansion of the 7TM bundle upon binding of MP1104 and Nb39 versus JDTic, with particularly pronounced movements in TM5–7 and ICL2. Distances were measured between the Cα atoms of K254^5.66, D266^6.27, Y330^7.53 and D168^ICL2. Nb39 and T4L fusion proteins have been omitted for clarity. (D) Reduction of orthosteric site volumes in KOP and MOP upon activation. (left) Superimposed pockets for KOP, inactive (PDB ID: 4DJH, grey) 1049 ų, and active (blue) 945 ų. (right) Superimposed pockets for MOP, inactive (PDB ID: 4DKL, grey) 1112 ų and active (PDB ID: 5C1M, green) 1053 ų. Volumes were calculated for the pockets of 4 superimposed receptors, and uniformly delimited between the level of extracellular lipid layer boundary (as predicted by OMP database), and the Cα atom of conserved residue W^6.48. See also Figure S2.

Figure 3. MP1104 interactions in the active state KOP binding pocket

(A) Binding pose comparison of MP1104 (orange) in the active state KOP (blue) compared with JDTic (purple) in the inactive state (grey, PDB ID: 4DJH). Main interactions involved MP1104 and binding pocket residues are shown, with hydrogen bonds depicted as dashed lines (black). (B) Comparison of MP1104 or SalA binding affinity at KOP wt and KOP D138^3.32A mutant using ³H-diprenorphine (N = 3). See Table S2 for values. (C) (top) Major interactions between the cyclopropylmethyl group of MP1104 (orange) and the hydrophobic pocket of active KOP (blue). (bottom) Comparison of binding pose between MP1104 (orange) and BU-72 (green) in KOP and MOP shows that MP1104 extends into the hydrophobic pocket but BU-72 does not. (D, E) Mutations of hydrophobic pocket residues (W287^6.48L, G319^7.42L and Y320^7.43L) strongly affect MP1104’s G protein activation (D) and β-arrestin2 recruitment (E), as measured by cAMP inhibition and Tango assay, respectively (N = 3). See Table S3, S4 for values See also Figure S3.

Figure 4. Activation signal propagation within KOP motifs

(A) Close ups highlight activation-related conformational changes in key receptor motifs, and show connection of structural changes from orthosteric site to the cytoplasmic transducer binding site. Conformational changes between active KOP (blue) and inactive KOP (grey) are highlighted for (B) MP1104 binding pocket, (C) P-I-F motif, (D) sodium binding pocket, (E) NPxxY motif, and (F) DRY motif. (G) KOP N141^3.35A mutation switches classic opioid receptor antagonists (left) into full agonists (right) in Tango-arrestin recruitment (N = 3). In KOP wt: MP1104 (red), EC₅₀ = 0.071±0.008 nM, E_max = 100±2; in KOP N141^3.35A: MP1104 (red), EC₅₀ = 0.027±0.008 nM, E_max = 100±2, naltrexone (orange), EC₅₀ = 6.53±0.90 nM, E_max = 107±3, naloxone (light green), EC₅₀ = 12.75±1.50 nM, E_max = 93±2, 6β-naltrexol (green), EC₅₀ = 22.47±1.8 nM, E_max = 120±2.

Figure 5. Structural insights for the design of biased and selective KOP ligands

(A) Chemical structures of MP1104 and IBNtxA. Chemical differences are highlighted in color. Comparison of iodobenzamide binding pose between MP1104 (orange, top) and docked IBNtxA (white, bottom) in KOP (blue). Y312^7.35 in KOP forms a hydrogen bond with amide oxygen of both compounds. (B) MP1104 (red) and IBNtxA (orange) are balanced full agonists in KOP measuring in cAMP inhibition and Tango-arrestin recruitment. Gi: MP1104, EC₅₀ = 0.003±0.001 nM, E_max = 97±1; IBNtxA, EC₅₀ = 0.002±0.001 nM, E_max = 96±1. Arrestin: MP1104, EC₅₀ = 0.035±0.010 nM, E_max = 115±8; IBNtxA, EC₅₀ = 0.032±0.010 nM, E_max = 113±12. Bias factor toward G protein: 0.6 and 1.5 for MP1104 and IBNtxA, respectively (N = 3). (C) IBNtxA (orange) displays G protein biased activity in MOP, while MP1104 (red) appears balanced in cAMP inhibition and Tango-arrestin recruitment. Gi: MP1104, EC₅₀ = 0.04±0.010 nM, E_max = 103±6; IBNtxA, EC₅₀ = 0.056±0.012 nM, E_max = 99±6. Arrestin: MP1104, EC₅₀ = 0.55±0.03 nM, E_max = 126±7; IBNtxA, EC₅₀ = 0.10±0.03 nM, E_max = 23±4. Bias factor toward G protein: 1.6 and 12 for MP1104 and IBNtxA, respectively (N = 3). (D) The KOP Y312^7.35W mutant (orange) shows slightly reduced MP1104- and strongly reduced IBNtxA-mediated arrestin recruitment compared to KOP wt (red) (N = 3). MP1104/KOP wt: EC₅₀ = 0.035±0.010 nM, E_max = 119±9; MP1104/KOP Y312^7.35W: EC₅₀ = 0.055±0.02 nM, E_max = 111±4. IBNtxA/KOP wt: EC₅₀ = 0.03±0.01 nM, E_max = 112±10; IBNtxA/KOP Y312^7.35W: EC₅₀ = 0.20±0.06 nM, E_max = 52±4. (E) Binding affinity of Nalfurafine (Ki = 0.32±0.02 nM in KOP and 4.20±0.21 nM in MOP) and compound 18 (Ki = 1.50±0.05 nM in KOP and 533±65 nM in MOP) (N = 3). See also Figure S4, S5.