Abstract
The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence–function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.
Highlights
IntroductionSmith 1970) in an attempt to settle the apparent contradiction between evolution by natural selection and the complex nature of the gene-encoded protein (Salisbury 1969)
The concept of 'protein space' was introduced in 1970 by John Maynard Smith (MaynardSmith 1970) in an attempt to settle the apparent contradiction between evolution by natural selection and the complex nature of the gene-encoded protein (Salisbury 1969)
The resulting tree built with Dop and PafA sequences indicated that Dop and PafA form two distinct and statistically well supported clusters that originated from an ancient duplication event (Fig. 2)
Summary
Smith 1970) in an attempt to settle the apparent contradiction between evolution by natural selection and the complex nature of the gene-encoded protein (Salisbury 1969). Proteins are of inherent restricted evolvability, as proteins are only marginally stable (ΔΔGunfolding ~ 5-10 kcal/mol) (DePristo et al 2005), and about one third of random mutations in proteins have severe effects on their function (>90% loss of activity) (Camps et al 2007). The mutational trajectory in which protein evolution occurs - while retaining catalytic activity and stability - is complex, given the stochastic nature of mutation and the vast sequence space of proteins. The effect of mutation is not additive and could be epistatic in nature; namely, the same mutation could be either neutral, beneficial or deleterious, depending on the context of the protein sequence. Interactions between mutations pose severe restrictions over evolutionary trajectories (Camps et al 2007; Kaltenbach and Tokuriki 2014)
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