The atomistic level structure for the activated human κ-opioid receptor bound to the full Gi protein and the MP1104 agonist

Abstract

The kappa opioid receptor (κOR) is an important target for pain therapeutics to reduce depression and other harmful side effects of existing medications. The analgesic activity is mediated by κOR signaling through the adenylyl cyclase-inhibitory family of Gi protein. Here, we report the three-dimensional (3D) structure for the active state of human κOR complexed with both heterotrimeric Gi protein and MP1104 agonist. This structure resulted from long molecular dynamics (MD) and metadynamics (metaMD) simulations starting from the 3.1-Å X-ray structure of κOR–MP1104 after replacing the nanobody with the activated Gi protein and from the 3.5-Å cryo-EM structure of μOR–Gi complex after replacing the 168 missing residues. Using MD and metaMD we discovered interactions to the Gi protein with strong anchors to two intracellular loops and transmembrane helix 6 of the κOR. These anchors strengthen the binding, contributing to a contraction in the binding pocket but an expansion in the cytoplasmic region of κOR to accommodate G protein. These remarkable changes in κOR structure reveal that the anchors are essential for activation.

Mechanism of β-arrestin recruitment by the μ-opioid G protein-coupled receptor

“…Activated State of Mouse μOR-DAMGO-Gi Complex. Strikingly, we found recently that ionic anchors are also essential for activation of Gi protein mediated by opioid receptors (15). Thus, in the presence of nonbiased agonists, we find a similar pattern in binding of Gi protein and βarr2 to the μOR.…”

“…Optimizing the cryo-EM structure (Fig. 3) of the active state of mouse-μOR bound to DAMGO stabilized by Gi protein (15,30) shows that ICL2 of μOR also involves extensive polar interactions with the Gi protein in which the salt bridge from D177 ICL2 (equivalent to the human D179 ICL2 ) to R32 on the Gαi subunit ( Fig. 3B) serves as an ionic anchor that engages Gi protein recruitment.…”

Effects of N-Substituents on the Functional Activities of Naltrindole Derivatives for the δ Opioid Receptor: Synthesis and Evaluation of Sulfonamide Derivatives

“…Indeed, co-crystals of opioid receptors with specific antagonists or agonists have been resolved to reveal the interaction between the protonated nitrogen atom in the ligands and the aspartic acid residue in the receptor [ 31 , 32 , 33 , 34 , 35 ]. Moreover, such interaction between a ligand and the receptor has also been recently elucidated by cryo-electron microscopic analysis of the ligand-receptor complex [ 36 , 37 ]. The neoclerodane diterpene salvinolin A is known to be a non-nitrogenous KOR agonist [ 38 ].…”

The quest for high-resolution G protein-coupled receptor–G protein structures

“…GPCRs are highly dynamic entities, and thus, determination of high-resolution structures of GPCRs in complex with their binding partners by X-ray crystallography or by cryoelectron microscopy (cryo-EM) techniques is a delicate experimental undertaking despite tremendous progress in recent years (3). In this issue of PNAS, Mafi et al (4) explore an alternative approach, molecular dynamics simulations, as a means to elucidate the atomistic structure of the human κ-opioid receptor (κOR) in complex with a potent agonist and the heterotrimeric Gi protein. To obtain structural information of GPCR-agonist-G protein complexes by computational methods is a welcome addition to existing experimental techniques.…”

“…β 2 AR, β 2 -adrenergic receptor; mini-G, minimal G protein; PDB, Protein Data Bank. *Engineered nanobody 35. engages in extensive contacts with the receptor, stabilizing its active conformation (4). The validity of the molecular dynamics simulations approach taken by Mafi et al (4) is assessed by simulating the κOR-nanobody crystal structure (17), building a simulated active-state μOR-Gi model from the μOR-nanobody crystal structure (18) and optimizing the cryo-EM μOR-Gi structure (16).…”