10.1371/journal.pone.0068841 Arhamatoulaye Maïga Arhamatoulaye Maïga Jon Merlin Jon Merlin Elodie Marcon Elodie Marcon Céline Rouget Céline Rouget Maud Larregola Maud Larregola Bernard Gilquin Bernard Gilquin Carole Fruchart-Gaillard Carole Fruchart-Gaillard Evelyne Lajeunesse Evelyne Lajeunesse Charles Marchetti Charles Marchetti Alain Lorphelin Alain Lorphelin Laurent Bellanger Laurent Bellanger Roger J. Summers Roger J. Summers Dana S. Hutchinson Dana S. Hutchinson Bronwyn A. Evans Bronwyn A. Evans Denis Servent Denis Servent Nicolas Gilles Nicolas Gilles Orthosteric Binding of ρ-Da1a, a Natural Peptide of Snake Venom Interacting Selectively with the α<sub>1A</sub>-Adrenoceptor Public Library of Science 2013 Biochemistry Neurochemistry Neurochemicals proteins Protein chemistry protein structure Transmembrane proteins Biomacromolecule-ligand interactions neuroscience neurotransmitters chromatography Liquid chromatography Reversed-phase chromatography orthosteric binding peptide venom interacting selectively 2013-07-25 01:49:00 Dataset https://plos.figshare.com/articles/dataset/Orthosteric_Binding_of_Da1a_a_Natural_Peptide_of_Snake_Venom_Interacting_Selectively_with_the_sub_1A_sub_Adrenoceptor/754857 <div><p>ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α<sub>1A</sub>-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of <sup>3</sup>H-prazosin or <sup>125</sup>I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α<sub>1A</sub>-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α<sub>1A</sub>-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α<sub>1A</sub>-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of <sup>3</sup>H-prazosin or <sup>125</sup>I-HEAT, and the IC<sub>50</sub> of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca<sup>2+</sup> release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α<sub>1A</sub>-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106<sup>3.32</sup>A and the S188<sup>5.42</sup>A/S192<sup>5.46</sup>A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86<sup>2.64</sup>A, F288<sup>6.51</sup>A and F312<sup>7.39</sup>A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86<sup>2.64</sup> was identified as a key interaction point for <sup>125</sup>I-HEAT, as the variant F86<sup>2.64</sup>A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α<sub>1A</sub>-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86<sup>2.64</sup>, F288<sup>6.51</sup> and F312<sup>7.39</sup>) and agonist (F288<sup>6.51</sup> and F312<sup>7.39</sup>) ligands. Its selectivity for the α<sub>1A</sub>-adrenoceptor may result, at least partly, from its interaction with the residue F86<sup>2.64</sup>, which appears to be important also for HEAT binding.</p></div>