Orthosteric binding of rho-Da1a, a natural peptide of snake venom interacting selectively with the alpha(1A)-Adrenoceptor

Arhamatoulaye Maiga, Jon Merlin, Elodie Marcon, Celine Rouget, Maud Larregola, Bernard Gilquin, Carole Fruchart-Gaillard, Evelyn Lajeunesse, Charles Marchetti, Alain Lorphelin, Laurent Bellanger, Roger James Summers, Dana Sabine Hutchinson, Bronwyn Anne Evans, Denis Servent, Nicolas Gilles

Research output: Contribution to journalArticleResearchpeer-review

Abstract

?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the a1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of 3H-prazosin or 125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the a1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the a1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the a1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the a1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D1063.32A and the S1885.42A/S1925.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F862.64A, F2886.51A and F3127.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F862.64 was identified as a key interaction point for 125I-HEAT, as the variant F862.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human a1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F862.64, F2886.51 and F3127.39) and agonist (F2886.51 and F3127.39) ligands. Its selectivity for the a1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding. ? 2013 Maiga et al.
Original languageEnglish
Article numbere68841
Number of pages11
JournalPLoS ONE
Volume8
Issue number7
DOIs
Publication statusPublished - 2013

Cite this

Maiga, Arhamatoulaye ; Merlin, Jon ; Marcon, Elodie ; Rouget, Celine ; Larregola, Maud ; Gilquin, Bernard ; Fruchart-Gaillard, Carole ; Lajeunesse, Evelyn ; Marchetti, Charles ; Lorphelin, Alain ; Bellanger, Laurent ; Summers, Roger James ; Hutchinson, Dana Sabine ; Evans, Bronwyn Anne ; Servent, Denis ; Gilles, Nicolas. / Orthosteric binding of rho-Da1a, a natural peptide of snake venom interacting selectively with the alpha(1A)-Adrenoceptor. In: PLoS ONE. 2013 ; Vol. 8, No. 7.
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title = "Orthosteric binding of rho-Da1a, a natural peptide of snake venom interacting selectively with the alpha(1A)-Adrenoceptor",
abstract = "?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the a1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of 3H-prazosin or 125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the a1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the a1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the a1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the a1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D1063.32A and the S1885.42A/S1925.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F862.64A, F2886.51A and F3127.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F862.64 was identified as a key interaction point for 125I-HEAT, as the variant F862.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human a1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F862.64, F2886.51 and F3127.39) and agonist (F2886.51 and F3127.39) ligands. Its selectivity for the a1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding. ? 2013 Maiga et al.",
author = "Arhamatoulaye Maiga and Jon Merlin and Elodie Marcon and Celine Rouget and Maud Larregola and Bernard Gilquin and Carole Fruchart-Gaillard and Evelyn Lajeunesse and Charles Marchetti and Alain Lorphelin and Laurent Bellanger and Summers, {Roger James} and Hutchinson, {Dana Sabine} and Evans, {Bronwyn Anne} and Denis Servent and Nicolas Gilles",
year = "2013",
doi = "10.1371/journal.pone.0068841",
language = "English",
volume = "8",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "7",

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Maiga, A, Merlin, J, Marcon, E, Rouget, C, Larregola, M, Gilquin, B, Fruchart-Gaillard, C, Lajeunesse, E, Marchetti, C, Lorphelin, A, Bellanger, L, Summers, RJ, Hutchinson, DS, Evans, BA, Servent, D & Gilles, N 2013, 'Orthosteric binding of rho-Da1a, a natural peptide of snake venom interacting selectively with the alpha(1A)-Adrenoceptor', PLoS ONE, vol. 8, no. 7, e68841. https://doi.org/10.1371/journal.pone.0068841

Orthosteric binding of rho-Da1a, a natural peptide of snake venom interacting selectively with the alpha(1A)-Adrenoceptor. / Maiga, Arhamatoulaye; Merlin, Jon; Marcon, Elodie; Rouget, Celine; Larregola, Maud; Gilquin, Bernard; Fruchart-Gaillard, Carole; Lajeunesse, Evelyn; Marchetti, Charles; Lorphelin, Alain; Bellanger, Laurent; Summers, Roger James; Hutchinson, Dana Sabine; Evans, Bronwyn Anne; Servent, Denis; Gilles, Nicolas.

In: PLoS ONE, Vol. 8, No. 7, e68841, 2013.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Orthosteric binding of rho-Da1a, a natural peptide of snake venom interacting selectively with the alpha(1A)-Adrenoceptor

AU - Maiga, Arhamatoulaye

AU - Merlin, Jon

AU - Marcon, Elodie

AU - Rouget, Celine

AU - Larregola, Maud

AU - Gilquin, Bernard

AU - Fruchart-Gaillard, Carole

AU - Lajeunesse, Evelyn

AU - Marchetti, Charles

AU - Lorphelin, Alain

AU - Bellanger, Laurent

AU - Summers, Roger James

AU - Hutchinson, Dana Sabine

AU - Evans, Bronwyn Anne

AU - Servent, Denis

AU - Gilles, Nicolas

PY - 2013

Y1 - 2013

N2 - ?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the a1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of 3H-prazosin or 125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the a1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the a1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the a1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the a1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D1063.32A and the S1885.42A/S1925.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F862.64A, F2886.51A and F3127.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F862.64 was identified as a key interaction point for 125I-HEAT, as the variant F862.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human a1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F862.64, F2886.51 and F3127.39) and agonist (F2886.51 and F3127.39) ligands. Its selectivity for the a1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding. ? 2013 Maiga et al.

AB - ?-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the a1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of 3H-prazosin or 125I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the a1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ?-Da1a for the a1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ?-Da1a, prazosin and HEAT compete at the a1A-adrenoceptor. ?-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 of ?-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ?-Da1a remained stable. The effect of ?-Da1a on agonist-stimulated Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the a1A-adrenoceptor were evaluated for alterations in ?-Da1a affinity. The D1063.32A and the S1885.42A/S1925.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F862.64A, F2886.51A and F3127.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F862.64 was identified as a key interaction point for 125I-HEAT, as the variant F862.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ?-Da1a interacts with the human a1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F862.64, F2886.51 and F3127.39) and agonist (F2886.51 and F3127.39) ligands. Its selectivity for the a1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding. ? 2013 Maiga et al.

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U2 - 10.1371/journal.pone.0068841

DO - 10.1371/journal.pone.0068841

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JO - PLoS ONE

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