Structural bases for stability-function tradeoffs in antibiotic resistance

Abstract

Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization "strain" of the active site. How this stability-activity tradeoff 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 beta-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of beta-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C beta-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol(-)(1) to 4.1 kcal mol(-)(1)), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 A from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. 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.

Figure 1

Beta-lactamase reaction mechanism and representative antibiotic substrates. (a) Reaction mechanism of AmpC beta-lactamase. (b) The first-generation cephalosporin antibiotic cephalothin. (c) The third-generation cephalosporin antibiotic cefotaxime.

Figure 2

ESBL substitutions under investigation in AmpC beta-lactamase, shown in purple. The catalytic serine is shown in green.

Figure 3

Relative enzyme activities and thermodynamic stabilities of AmpC ESBL mutants. (a) Relative activities of AmpC ESBLs against the first-generation cephalosporin cephalothin (blue) and the third-generation cephalosporin cefotaxime (purple). (b) Characteristic thermal denaturation curves of selected AmpC ESBL mutants (V298E = orange, Omega loop insertion = green, T70I = magenta) relative to the wild-type enzyme (blue) (c) The differential stabilities of AmpC ESBL mutants relative to wild-type AmpC.

Figure 4

The x-ray crystal structure of V298E to 2.6 Å resolution, showing an overview of changes in the V298E mutant (purple) compared to the WT protein (green). Density is lost for the region of the loop shown in red, presumed to have flipped out. Structural changes are shown at the point of mutation (inset), with the mutant protein (purple) overlaid on the WT protein (green).

Figure 5

The x-ray structure of the “omega loop insertion” mutant (H210AAA) to 1.6 Å resolution. (a) The omega loop insertion structure (orange, 3 alanine insertion in purple) overlaid on the WT protein (green). (b) F_o-F_c omit density for residues 210–216 shown at 3σ.

Figure 6

Flexibility induced in extended-spectrum mutants T70I and E219K. (a) The T70I apo structure (purple) overlaid on the WT structure (green). Density is lost from residues 193–221 in the omega loop (shown in transparent green) in the T70I structure. (b) The T70I/benzo(b)thiophene (BZB) structure (purple) overlaid on the WT structure (green). Residues 193–221, lost in the T70I structure but present in the T70I/benzo(b)thiophene structure, are shown in cartoon. (c) Stereo view of the x-ray crystal structure of E219K/BZB to 1.63 Å resolution. F_o-F_c omit density is shown at 3σ. Arrow highlights the two conformations of residues 215–216, distinguished by a peptide flip. (d) The omega loop and active site of E219K/BZB (orange) overlaid with that of the wild-type AmpC structure (green), showing the conformational differences in the 211–213 region. The position of BZB is shown in the active site.

Figure 7

X-ray structure of Y221G/cefotaxime (2.3 Å). (a) Stereo view of quality of density of cefotaxime in the active site of Y221G. 2F_o-F_c density (blue) is shown at a contour level of 1σ, and F_o-F_c.omit density (green) is shown at a contour level of 3σ. (b) The Y221G/cefotaxime structure (orange) overlaid with the WT/ceftazidime structure (green). (c) The Y221G/cefotaxime structure (orange) overlaid with the WT/loracarbef structure (purple). (d) The Y221G/cefotaxime structure (orange) overlaid with the WT/ceftazidime deacylation transition state analog (gray).