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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 ?-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 ?1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three ?2 adrenoceptor subtypes. Three positions in the ?-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the ?1 and ?2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

SUBMITTER: Blanchet G

PROVIDER: S-EPMC5457417 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins.

Blanchet Guillaume G Alili Doria D Protte Adèle A Upert Gregory G Gilles Nicolas N Tepshi Livia L Stura Enrico A EA Mourier Gilles G Servent Denis D

Scientific reports 20170602 1

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 fu ...[more]

PMID: 28578406

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Project description:BackgroundIn addition to providing phylogenetic relationships, tree making procedures such as parsimony and maximum likelihood can make specific predictions of actual historical sequences. Resurrection of such sequences can be used to understand early events in evolution. In the case of RNA, the nature of parsimony is such that when applied to multiple RNA sequences it typically predicts ancestral sequences that satisfy the base pairing constraints associated with secondary structure. The case for such sequences being actual ancestors is greatly improved, if they can be shown to be biologically functional.ResultsA unique common ancestral sequence of 28 Vibrio 5S ribosomal RNA sequences predicted by parsimony was resurrected and found to be functional in the context of the E. coli cellular environment. The functionality of various point variants and intermediates that were constructed as part of the resurrection were examined in detail. When separately introduced the changes at single stranded positions and individual double variants at base-paired positions were also viable. An additional double variant was examined at a different base-paired position and it was also valid.ConclusionsThe results show that at least in the case of the 5S rRNAs considered here, ancestors predicted by parsimony are likely to be realistic when the prediction is not overly influenced by single outliers. It is especially noteworthy that the phenotype of the predicted ancestors could be anticipated as a cumulative consequence of the phenotypes of the individual variants that comprised them. Thus, point mutation data is potentially useful in evaluating the reasonableness of ancestral sequences predicted by parsimony or other methods. The results also suggest that in the absence of significant tertiary structure constraints double variants that preserve pairing in stem regions will typically be accepted. Overall, the results suggest that it will be feasible to resurrect additional meaningful 5S rRNA ancestors as well as ancestral sequences of many different types of RNA.

Dataset Information

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins.

Publications

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins.

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