Structural Bases for Stability–Function Tradeoffs in Antibiotic Resistance

Abstract

Pre-organization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein, and consequently enzymes can be dramatically stabilized by substitutions that attenuate the size and pre-organization “strain” of the active site. How this stability-activity trade-off constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities, or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100- to 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability – from 1.7 to 4.1 kcal/mol – consistent with the function-stability hypothesis. Intriguingly, several of the substitutions were 10 – 20 Å from the catalytic serine; the question arose how they conferred extended-spectrum activity. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by x-ray crystallography. Distinct mechanisms of action are revealed for each mutant, including changes in the flexibility and ground state structures of the enzyme. These results explain the structural bases for the antibiotic resistance conferred by these substitutions, and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.

Stability-activity tradeoffs constrain the adaptive evolution of RubisCO

“…These mutations tend to be destabilizing and, on the basis of structural context and earlier mutational studies of algal RubisCOs, are inferred to modify the active site loop dynamics or position of residues at the P5 and O 2 /CO 2 binding sites. Whereas adaptive mutations 10-20 Å from active sites have occasionally been identified in other enzymes (31), in RubisCO, these form the majority of positively selected sites that distinguish C 3 and C 4 species. Experiments with RubisCO from the green alga Chlamydomonas reinhardii previously implicated the interfaces between large and small subunits in the modulation of catalytic rates (32).…”

Characterization of the Global Stabilizing Substitution A77V and Its Role in the Evolution of CTX-M β-Lactamases

“…These enzymes arise due to mutations resulting in substitutions near the active site that increase hydrolysis of oxyimino-cephalosporins such as cefotaxime and ceftazidime. The substitutions that increase hydrolysis and create new interactions with the substrate or transition state often remove interactions that would otherwise stabilize the enzyme in the absence of substrate (3,5,12,13). These variant enzymes depend strongly on stabilizing mutations that correct the stability defect associated with the original mutations (12,14).…”

“…The substitutions that increase hydrolysis and create new interactions with the substrate or transition state often remove interactions that would otherwise stabilize the enzyme in the absence of substrate (3,5,12,13). These variant enzymes depend strongly on stabilizing mutations that correct the stability defect associated with the original mutations (12,14). For example, the M182T mutation has been identified in many TEM-1 ␤-lactamase variants from clinical isolates, and it compensates for stability and folding defects caused by various mutations in the enzyme that increase oxyimino-cephalosporin hydrolysis (15).…”

Evolution of Broad Spectrum β-Lactam Resistance in an Engineered Metallo-β-lactamase

“…However, we were unable to purify the wild type evMBL9 protein, although several attempts were made using different strategies, indicating a high instability of this engineered protein. Directed evolution studies carried out on serine-␤-lactamases have shown that resistance mutations compromise the protein stability and are usually followed by compensatory mutations that restore the stability (35)(36)(37)(38). Instead of directly measuring the thermal stability of the evMBL9 variants, the protein/mRNA ratios were determined for the resistant transformants to infer the protein stability.…”