Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

Abstract

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α 1A -adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α 1 and α 2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.Evolutionary processes of venomous animals have selected enzymes and disulfide-rich peptides in their venoms to improve their ability to subdue their prey and defend against predators. These compounds act predominantly on few, well characterized and physiologically-relevant molecular targets of the envenomed animals 1 . Recruited by convergent evolution, toxins impact mainly the haemostatic, nervous and cardiovascular systems 2 . Some toxins present in venoms, in spite of their debilitating effects, have become live-saving drugs [3][4][5] . To exert their biological activities, toxins interact mainly with ion channels, coagulation factors, nicotinic receptors, cell membrane or enzymes but only rarely with the most important class of physiological targets, i.e, the G protein-coupled receptors (GPCRs) 6 . Compared to numerous toxins that act on voltage-gated or ligand-gated ion channels, only a few toxins that interact with GPCRs have been identified. These toxins have been isolated mainly from cone snails venoms such as conopressin interacting with vasopressin receptor 7 , contulakin-G which binds to neurotensin receptor 8 , ρ-conotoxin TIA specific of the α 1A -adrenoceptor 9 and the τ-conotoxin CnVA that interacts with the somatostatin sst3 receptor 10 . The black widow spider and the gila monster provide further examples of GPCRs interacting toxins, namely, α-latrotoxin interacting with the latrophilin receptor 11 and exenatide, exploited as an anti-diabetic drug 12 , that targets the GLP-1 receptor, respectively. In addition, mamba venoms contain aminergic toxins that recognize various bioaminergic receptors [13][14][15][16][17][18][19] . Aminergic toxins belong to the three-finger fold toxin (3FT) superfamily, a structural fold known to support a large diversity of...