Multiple mutations
often have non-additive (epistatic) phenotypic
effects. Epistasis is of fundamental biological relevance but is not
well understood mechanistically. Adaptive evolution, i.e., the evolution
of new biochemical activities, is rich in epistatic interactions.
To better understand the principles underlying epistasis during genetic
adaptation, we studied the evolution of TEM-1 β-lactamase variants
exhibiting cefotaxime resistance. We report the collection of a library
of 487 observed evolutionary trajectories for TEM-1 and determine
the epistasis status based on cefotaxime resistance phenotype for
206 combinations of 2–3 TEM-1 mutations involving 17 positions
under adaptive selective pressure. Gain-of-function (GOF) mutations
are gatekeepers for adaptation. To see if GOF phenotypes can be inferred
based solely on sequence data, we calculated the enrichment of GOF
mutations in the different categories of epistatic pairs. Our results
suggest that this is possible because GOF mutations are particularly
enriched in sign and reciprocal sign epistasis, which leave a major
imprint on the sequence space accessible to evolution. We also used
FoldX to explore the relationship between thermodynamic stability
and epistasis. We found that mutations in observed evolutionary trajectories
tend to destabilize the folded structure of the protein, albeit their
cumulative effects are consistently below the protein’s free
energy of folding. The destabilizing effect is stronger for epistatic
pairs, suggesting that modest or local alterations in folding stability
can modulate catalysis. Finally, we report a significant relationship
between epistasis and the degree to which two protein positions are
structurally and dynamically coupled, even in the absence of ligand.