Identification of the Molecular Mechanisms by Which the Diterpenoid Salvinorin A Binds to -Opioid Receptors
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- 作者：Feng Yan ; Philip D. Mosier ; Richard B. Westkaemper ; Jeremy Stewart ; Jordan K. Zjawiony ; Timothy A. Vortherms ; Douglas J. Sheffler ; and Bryan L. Roth
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 21, 2005
- 年：2005
- 卷：44
- 期：24
- 页码：8643 - 8651
- 全文大小：534K
- 年卷期：v.44,no.24(June 21, 2005)
- ISSN：1520-4995
文摘
Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates 
-opioid receptors (KORs). Salvinorin A is unique in that it is theonly 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-nitrogenousopioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high bindingaffinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilizedin 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 tyrosinesTyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KORagonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differentialrequirements 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 andan analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to bein close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showedenhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that thediterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectivelyactivate KORs.
-opioid receptors (KORs). Salvinorin A is unique in that it is theonly 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-nitrogenousopioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high bindingaffinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilizedin 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 tyrosinesTyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KORagonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differentialrequirements 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 andan analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to bein close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showedenhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that thediterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectivelyactivate KORs.