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>