PDBsum entry 1ozc

Signaling protein

PDB id: 1ozc

Contents

Protein chain

268 a.a.

References listed in PDB file

Key reference

Title

Opioid receptor random mutagenesis reveals a mechanism for g protein-Coupled receptor activation.

Authors

F.M.Décaillot, K.Befort, D.Filliol, S.Yue, P.Walker, B.L.Kieffer.

Ref.

Nat Struct Biol, 2003, 10, 629-636. [DOI no: 10.1038/nsb950]

PubMed id

12847517

Abstract

The high resolution structure of rhodopsin has greatly enhanced current understanding of G protein-coupled receptor (GPCR) structure in the off-state, but the activation process remains to be clarified. We investigated molecular mechanisms of delta-opioid receptor activation without a preconceived structural hypothesis. Using random mutagenesis of the entire receptor, we identified 30 activating point mutations. Three-dimensional modeling revealed an activation path originating from the third extracellular loop and propagating through tightly packed helices III, VI and VII down to a VI-VII cytoplasmic switch. N- and C-terminal determinants also influence receptor activity. Findings for this therapeutically important receptor may apply to other GPCRs that respond to diffusible ligands.

Figure 3.
Figure 3. Locations of activating mutations. (a) Mutated amino acids are highlighted on a schematic representation of the human -opioid receptor sequence using single-letter amino acid codes. N- and C-terminal mutated amino acids as well as Phe159, Pro182 and Thr213 are shown in black circles. Mutated amino acids of groups 1, 2, 3 and 4 are highlighted in orange, green, yellow and blue, respectively. Mutated residues that are part of well-known GPCR structural motifs are Trp173, Pro182, Trp274, Tyr318 and Arg258 and mutated residues that are moderately to well conserved are Lys214, Met262, Val283, Leu286, Tyr308 and Glu323. To orient our mutated residues relative to well described, common class-A GPCR amino acid residues, a white square indicates one of the most conserved residues in each helix: Asn67 (hI), Asp95 (hII), Arg146 (hIII), Trp173 (hIV), Pro225 (hV), Pro276 (hVI) and Pro315 (hVII). (b) Except for the residues indicated in black, the mutated residues were modeled and spatially clustered into four groups. Position of modeled residues is indicated on a three-dimensional model (PDB accession code 1OZC) with side chains shown in yellow.

Figure 5.
Figure 5. Mechanism for -opioid receptor activation by an agonist. Schematic of receptor showing a series of activation events proposed from our analysis of CAM delta receptors. Postulated helical movements are indicated by arrows. Step A: the opioid agonist (black bar) binds to e3 and possibly to N-terminal determinants. This perturbs the e3 hydrophobic cluster, thereby destabilizing hVI-hVII interactions of the helical bundle near the extracellular side of the receptor. Step B: the amphiphilic agonist enters the binding pocket, disrupting both hydrophobic (hIII-hVI) and hydrophilic (hIII-hVII) interactions and provoking outward hIII-hVI-hVII movements. hIII moves toward the hII-hIV interface while hVI and hVII separate from each other. Step C: helical movements propagate downward within the receptor, break cytoplasmic ionic locks (hVI-hVII from our study and perhaps hIII-hVI; see text) and possibly release putative hVIII. This reveals receptor intracellular determinants that interact better with G proteins^52. Mutations from group 1 mimic step A, mutations from groups 2 and 3 simulate events in step B, and group 4 mutations produce structural modifications as in step C.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2003, 10, 629-636) copyright 2003.