title: Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors.
abstract: Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.
authors.affiliation: Department of Biochemistry, Case Western Reserve University Medical School, Cleveland, Ohio 44106, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Yan
authors.forename: Philip D
authors.initials: PD
authors.lastname: Mosier
authors.forename: Richard B
authors.initials: RB
authors.lastname: Westkaemper
authors.forename: Jeremy
authors.initials: J
authors.lastname: Stewart
authors.forename: Jordan K
authors.initials: JK
authors.lastname: Zjawiony
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Vortherms
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Sheffler
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Roth
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018076:DNA, Complementary; D004224:Diterpenes / Q000737:chemistry*; D045785:Diterpenes, Clerodane; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017473:Receptors, Opioid, kappa / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D027544:Salvia
chemical_list: D018076:DNA, Complementary; D004224:Diterpenes; D045785:Diterpenes, Clerodane; D017473:Receptors, Opioid, kappa; D011994:Recombinant Proteins; C090499:salvinorin A