Negative Epistasis and Evolvability in TEM-1 β-Lactamase—The Thin Line between an Enzyme's Conformational Freedom and Disorder

Abstract

Epistasis is a key factor in evolution, since it determines which combinations of mutations provide adaptive solutions and which mutational pathways towards these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis – a complete reversion of mutational effects, particularly in protein evolution, yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in the antibiotic-resistance enzyme TEM-1 β-lactamase. Separated by 10Å, these mutations initiate two separate trajectories towards increased hydrolysis rates and resistance towards second and third-cephalosporins antibiotics. Both mutations allow the enzyme’s active-site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures we found that whereas G238S induces discrete conformations, R164S causes local disorder. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme’s catalytic pre-organization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing, global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of proteins structures and functions, but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.

Modelling proteins’ hidden conformations to predict antibiotic resistance

“…This hypothesis is supported by computational models and room temperature crystals that have revealed TEM adopts diverse structures11121314. A growing body of work argues for the importance of conformational heterogeneity in processes like allostery151617, ligand binding1819202122 and catalysis42324.…”

Multitarget selection of catalytic antibodies with β‐lactamase activity using phage display

“…Studies have shown that these modifications effectively aid the enzyme resistance by expanding the active site cavity in order to either accommodate larger substrates or improve the formation of electrostatic bonds . Identifying the vast spectrum of structural motifs capable of holding this catalytic activity is therefore essential to understand the evolutionary capabilities of an enzyme and an important step toward the future design of clinically useful antibiotics and β‐lactamase inhibitors.…”

The Moderately Efficient Enzyme: Futile Encounters and Enzyme Floppiness

“…Specifically, Asn170 that coordinates the deacylating water molecule adopts a non--catalytic configuration, even when a covalent inhibitor occupies the active--site. The entropic cost of selecting from multiple sub--states, and foremost, the dominance of an impaired conformation of N170, underlie the very low catalytic efficiency of the double mutant(85).…”