“Biophysical and structural traits in protein evolution”

A. J. VILA

Buenos Aires

Conferencia; EMBL, Biología Molecular de Avanzada; 2012

European Molecular Biology Laboratory (EMBL)

Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given three dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by β-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-β-lactamase (MβL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by two mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of one metal ion; while the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MβLs have not exhausted their robustness threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thourough characterization of the selected proteins can be successfully employed to predict future evolutionary events and design inhibitors with an evolutionary perspective.