Orthosteric Binding of ρ-Da1a, a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

Abstract

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-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 α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-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 α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of 3H-prazosin or 125I-HEAT, and the IC50 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 Ca2+ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-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, ρ-Da1a interacts with the human α1A-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 α1A-adrenoceptor may result, at least partly, from its interaction with the residue F862.64, which appears to be important also for HEAT binding.

Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor

“…The presence of glycine in in EC1 of BB 2 receptor is essential for determining high affinity of human BnRs (BB 2 , BB 1 and BB 3 receptors) for the agonist, peptide #1 [D- Tyr 6 , βAla 11 ,Phe 13 ,Nle 14 ]Bn-(6–14)] [41] and a glycine in the TM1 of the ETA receptor [72] or TM6 of the melatonin receptor [73], is important for determining high affinity for endothelin and melatonin, respectively. In the AT1 receptor the presence of a glutamic acid in TM7 is essential for high affinity interaction with angiotensin [74] as is its presence in TM1 of MCR4 receptor needed for binding and full potency of the peptide agonist, JRH887–9 [75]. …”

Molecular exploration of the α1A‐adrenoceptor orthosteric site: Binding site definition for epinephrine, HEAT and prazosin

“…However, the study does not report the expression level of the mutant at membrane surface, excluding a clear explanation of this result. Our result indicate that D106A α 1A ‐AR receptor was very difficult to express with a B max around 0.6 pmol/mg, 30‐times lower as compared to the wild‐type receptor (Table 1, [10]). By homologous and heterologous binding experiments we unambiguously demonstrated that the D106A variant affect only epinephrine affinity, and in a moderate manner (5.3 times affinity decrease), with no change in prazosin or HEAT affinities.…”

“…We explored positions around the F86 (important position for HEAT affinity [10] and identified E87 and W313 as possible positions in interaction with HEAT. Both mutations E87A and W313A slightly affect by 4.8 and 4.5 times 125 I-HEAT affinity, without modifying neither prazosin nor epinephrine affinities.…”

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

“…7 ). The selectivity profile of AncTx1 for the α 1A subtype surpasses that of all modern and ancestral aminergic toxins (Figs 5 and 7 , Table 1 ) with a selectivity factor (the ratio between the affinity constants for the 2 most highly targeted receptors) that is 12-times better in AncTx1 compared to ρ-Da1a, currently the most selective peptide for the α 1A subtype 35 . The selectivity of AncTx1 is characterized by a slightly higher affinity for α 1A and a much lower one for the α 1B and α 2C receptors, relative to ρ-Da1a (Fig.…”