Local and Global Protein Interactions Contribute to Residue Entrenchment in Beta-Lactamase TEM-1

Abstract

Due to their rapid evolution and their impact on healthcare, beta-lactamases, protein degrading beta-lactam antibiotics, are used as generic models of protein evolution. Therefore, we investigated the mutation effects in two distant beta-lactamases, TEM-1 and CTX-M-15. Interestingly, we found a site with a complex pattern of genetic interactions. Mutation G251W in TEM-1 inactivates the protein's function, just as the reciprocal mutation, W251G, does in CTX-M-15. The phylogenetic analysis revealed that mutation G has been entrenched in TEM-1's background: while rarely observed throughout the phylogeny, it is essential in TEM-1. Using a rescue experiment, in the TEM-1 G251W mutant, we identified sites that alleviate the deviation from G to W. While few of these mutations could potentially involve local interactions, most of them were found on distant residues in the 3D structure. Many well-known mutations that have an impact on protein stability, such as M182T, were recovered. Our results therefore suggest that entrenchment of an amino acid may rely on diffuse interactions among multiple sites, with a major impact on protein stability.

Keywords: CTX-M-15 beta-lactamase; M182T mutation; TEM-1 beta-lactamase; entrenchment; protein stability.

Figure 1

(A) Effects of mutations on MIC score when changing TEM-1 residue with that of CTX-M-15 (n = 236, dataset from Firnberg et al. [8]). (B) Effects of mutations on MIC score when changing CTX-M-15 residue with that of TEM-1 (n = 25). MIC score corresponds to log₂(MIC_mutant/MIC_WT). For each collection, one single mutant harbored an inactivating mutation (MIC score = −5): G251W in TEM-1, and W251G in CTX-M-15.

Figure 2

Phylogenetic tree of 157 class A beta-lactamases [9]. The different colors indicate the diversity at position 251. Beta-lactamases with a G251 are labeled in black, and TEM-1 and CTX-M-15 beta-lactamases are labeled in red.

Figure 3

(a) Three-dimensional structure of TEM-1 (pdb entry 1BTL). The site G251 is shown in pink, and the 3 residues exclusively associated with G251 (E48, R259, and W290) are indicated in blue. (b) Three-dimensional structure of CTX-M-15 (pdb entry 4HBT). The site W251 is shown in pink, and the 3 residues associated with W251 (L48, L259, and L290) are indicated in blue.

Figure 4

Selective mutational effect of mutations in the 3 amino acids found exclusively in the G251 group (n = 8) (E48, R259, and W290). Fitness was obtained by using Firnberg’s data on TEM-1 [8]. A fitness of 1 (green) corresponds to the fitness of the wild-type allele (TEM-1). A fitness of 0 (red) corresponds to a minimum fitness. The color gradient from green to red corresponds to the fitness gradient. The boxed mutations correspond to amino acids present in CTX-M-15.

Figure 5

Two opposite faces of the protein (a,b). Sites with compensating mutations on G251W-TEM variant (pdb entry: 1 BTL) are indicated with different colors. The active site (S70) is indicated in green and site 251 in pink. Mutations with local effects (<13 Å from W251) are indicated in yellow, and those with a global effect are in red. Mutations located <13 Å from W251 but previously described with a global stabilizing effect on the protein are indicated in orange.

Figure 6

Steps of entrenchment of the residue G251 in TEM-1. The 3 sites (48, 259, and 290) that coevolve with G251 and two sites involved in compensating mutations (153 and 182) are represented beside site 251. The ability of each beta-lactamase to tolerate W251 or G251 is represented if it was validated experimentally.