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 α1A-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 α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.
Highlights
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
Aminergic toxins belong to the three-finger fold toxin (3FT) superfamily, a structural fold known to support a large diversity of biological functions[20]
Published phylogenetic analysis showed that the aminergic toxins are a functional family developed in the mamba venoms and that a high sequence identity is shared among them[19]
Summary
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. Interacts efficiently and non-selectively with seven different aminergic receptors (Fig. 1B) These observations suggest that during the evolution of these toxins various functional properties diverged, were modified and, the least useful ones for the snake, dispensed with. By constructing and testing function of these ancestral proteins and comparing the results with extant proteins, this method is able to follow amino acids changes in the evolutionary history and measure their impact on their properties This “evolutionary biochemistry”[27] open a new avenue for protein engineering[28] by creating small but functionally rich libraries[29]. As the first proof of concept for enzyme engineering, Gaucher and colleagues use the REAP (Reconstructing Evolutionary Adaptive Paths) to engineer new Taq polymerases with original properties[30, 31]
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