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Capturing the mutational landscape of the beta-lactamase TEM-1

  1. Olivier Tenaillona,b,1
  1. aInstitut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche en Santé (UMR-S) 722, F-75018 Paris, France;
  2. bUniv Paris Diderot, Sorbonne Paris Cité, UMR-S 722 INSERM, F-75018 Paris, France;
  3. cService de Bactériologie-Virologie, Groupe Hospitalier Lariboisière-Fernand Widal, Assistance Publique-Hôpitaux de Paris (AP-HP), F-75475 Paris, France;
  4. dINSERM, UMR-S 738, F-75018 Paris, France;
  5. eUniv Paris Diderot, Sorbonne Paris Cité, UMR-S 738 INSERM, F-75018 Paris, France;
  6. fLaboratoire de Biochimie, UMR 7654, Ecole Polytechnique, Centre National de la Recherche Scientifique, F-91128 Palaiseau Cedex, France;
  7. gEquipe d'Accueil 3964, Université Paris Diderot, F-75018 Paris, France;
  8. hGénoscope, Commissariat à L’Energie Atomique–Institut de Génomique, 91057 Evry Cedex, France;
  9. iService de Microbiologie-Hygiène, Hôpital Louis Mourier, AP-HP, F-92700 Colombes, France; and
  10. jCentre d’Ecologie Fonctionnelle et Evolutive, Centre National de la Recherche Scientifique, UMR5175, F-34293 Montpellier Cedex 5, France

  1. Edited by Bruce R. Levin, Emory University, Atlanta, GA, and approved June 25, 2013 (received for review September 21, 2012)

  1. Fig. 1.
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    Fig. 1.

    Distribution of mutation effects on the MIC to amoxicillin in mg/L. (A) For each amino acid along the protein, excluding the signal peptide, the average effect of mutations on MIC is presented in the gene box with a color code, and the effect of each individual amino acid change is presented above. The color code corresponds to the color used in B. Gray bars represent amino acid changes reachable through a single mutation that were not recovered in our mutant library. Amino acids considered in the extended active site are associated with a blue bar beneath the gene box. (B) Distribution of mutation effects on the MIC is presented in color bars (n = 990); white bars illustrate the distribution of MIC of the wild-type clones (n = 1,594), in other words the noise in MIC measurement. (C) Representation of the average effect of mutations on MIC for each residue on the 3D structure of the protein.

  2. Fig. 2.
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    Fig. 2.

    Determinants of mutations effects on MIC. (A) Average effect of amino acid changes on MIC is presented as a matrix. The color code is identical to the one in Fig. 1. (B) Matrix BLOSUM62, representing amino acid penalty used in protein alignments using a color gradient of the same range as in A. In both matrices, only amino acid changes observed in the mutant library are colored. (C) Impact of accessibility to the solvent on mutant’s MIC. The distribution of accessibility of amino acids (buried = 0, fully accessible = 100) is plotted for different categories of mutants sharing the same MIC. Large effect mutations are enriched for buried sites. (D) Impact of predicted effect of mutations on protein stability (∆∆G estimated by PopMusic software) on mutant’s MIC. The distribution of ∆∆G of mutants (∆∆G > 0 is destabilizing, ∆∆G < 0, stabilizing) is plotted for different categories of mutants sharing the same MIC. Large effect mutations are enriched for destabilizing mutations. In C and D, hatched fractions represent amino acids included in the active site. The color code is similar to that of Fig. 1.

  3. Fig. 3.
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    Fig. 3.

    Epistatic interactions due to the stabilizing mutation M182T. (A) Distribution of mutation effects on MIC in M182T, for mutants also found in the TEM-1 library (n = 167). The color of the bars represents the MIC in the TEM-1 background of the mutants. A much larger fraction of mutants with no effect on MIC is found in M182T and is composed of mutants found to have some deleterious effects in TEM-1 background. (B) Plot of the MIC score in the two different backgrounds. The size of dots represents the number of mutants in that spot. The large fraction of points in the upper diagonal illustrates the compensating effect of mutation M182T. (C and D) Observed (colored bars) and predicted (white bars) distributions of mutant MICs in TEM-1 (C) and M182T backgrounds (D), using a three-parameter biophysical model of stability and excluding the active site.

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