Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM β-lactamases

Abstract

The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function.

Fig. 1

Diagram illustrating the structure of TEM-1 β-lactamase and identifying the amino acid substitutions relevant to this study. A cartoon representation of the overall and secondary structural elements is shown in gray with the position of the catalytic serine (S70) marked by a blue ball in the TEM-1 active site. The destabilizing substitutions and second-site suppressors examined in this study are shown as red and green sticks, respectively, with the mesh outlining the van der waals radius of the atoms. The right panel view of TEM-1 (a) is an 180° degree rotation of the left (b). The amino acid positions are numbered according to the Ambler numbering scheme.

Fig. 2

Survival curves of E. coli containing the pBG66 plasmid encoding the TEM-1 β-lactamase H158S:V159S:T160H (158-60SSH) mutants and secondary suppressor substitutions. (a) Colony forming units (cfu) on agar plates containing increasing concentrations of ampicillin for E. coli containing 158-60SSH or I47V single mutants or the 158-60SSH:I47V double mutant. (b) Cfu for E. coli containing 158-60SSH or A184V single mutants or the double mutant. (c) Cfu for E. coli containing T188I mutant combinations. (d) Cfu for E. coli containing I208M mutant combinations. (e) Cfu for E. coli containing R241H mutant combinations. (f) Cfu for E. coli containing T265M mutant combinations. (g) Cfu for E. coli containing R275Q mutant combinations. (h) Cfu for E. coli containing N276D mutant combinations.

Fig. 3

Survival curves of E. coli with pBG66 plasmid encoding TEM-1 β-lactamase L76N mutants and secondary suppressor substitutions. (a) Colony forming units (cfu) on agar plates containing increasing concentrations of ampicillin for E. coli containing L76N or I47V single mutants or the L76N: I47V double mutant. (b) Cfu for E. coli containing L76N and A184V mutant combinations as in (a). (c) Cfu for E. coli containing L76N and T188I mutant combinations. (d) Cfu for E. coli containing L76N and I208M mutant combinations. (e) Cfu for E. coli containing L76N and R241H mutant combinations. (f) Cfu for E. coli containing L76N and T265M mutant combinations. (g) Cfu for E. coli containing L76N and R275Q mutant combinations. (h) Cfu for E. coli containing L76N and N276D mutant combinations.

Fig. 4

Steady state protein expression levels of wild-type and mutant TEM-1 β-lactamases as determined by immunoblot analysis of whole cell lysates of E. coli cultures containing pBG66 plasmid encoding wild type or mutants. Lanes representing wild-type and mutants are labeled.

Fig. 5

Thermal denaturation curves of TEM-1 wild-type and selected β-lactamase variants. Fractional changes of the CD signal for the wild-type (black), M182T (red), H158S:V159S:T160H:M182T (green) and A184V (blue) β-lactamases are shown. The results are representative of the results for the other mutants which are shown in Figure S1. The T_m values (Table 4) were determined by fitting a sigmoidal function to the transition from the folded to unfolded state (see Materials and Methods).

Fig. 6

Diagrams of global suppressor substitutions in the TEM-1 structure. Superimposition of the wild-type (gray) and mutant (cyan) crystal or modeled structures are illustrated (see results). The local environment of the global suppressor substitutions (a) M182T, (b) A184V, (c) I208M, (d) T265M, (e) R275Q and (f) N276D are shown in comparison to wild-type (1XPB). The dashed black lines indicate hydrogen bonds and the red sphere are water molecules. The Protein Data Bank codes for the M182T, A184V, I208M, and N276D mutants are 1JWP, 1BTL, 3DTM, and 1CK3, respectively.