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Pervasive degeneracy and epistasis in a protein-protein interface

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Science  06 Feb 2015:
Vol. 347, Issue 6222, pp. 673-677
DOI: 10.1126/science.1257360
  • Fig. 1 Mapping sequence space.

    (A) PhoQ phosphorylates or dephosphorylates PhoP depending on extracellular magnesium concentration. White asterisks indicate interfacial residues randomized in the phoQ library. (B) YFP levels measured by flow cytometry for cells expressing wild-type phoQ, lacking phoQ, or harboring the phoQ library, before and after selection. Shaded regions indicate wild-type YFP levels.

  • Fig. 2 Functional degeneracy of PhoQ interfacial residues.

    (A) Functionality of point mutations assessed individually. Blue indicates functional variants; magenta boxes indicate wild-type residues. Amino acid abbreviations: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (B) Flow cytometry measurements of YFP levels for 20 PhoQ variants. Error bars indicate SD; n = 2. (C) PhoQ variants indicated were autophosphorylated in vitro and tested for phosphotransfer to and dephosphorylation of PhoP. (D) Head-to-head competitions of wild-type against strains expressing the indicated PhoQ variant. (E) Summary of functional PhoQ variants. (F) Heat map indicating amino acid frequencies in the 1659 functional PhoQ variants.

  • Fig. 3 Epistasis of PhoQ interfacial residues.

    (A and B) Flow cytometry (A) and in vitro analysis (B) of the PhoQ variants indicated. Shaded regions indicate wild-type YFP levels. (C and D) Head-to-head competitions of the wild type against strains producing the indicated PhoQ variant. (E) Venn diagram comparing the number of functional PhoQ variants identified with that predicted from single mutants, assuming position independence. (F) Heat map showing frequency of residue pairs at positions 1 and 3 relative to frequency expected if residues occurred independently. (G) Frequency logos for residues at each position in the PhoQ sets indicated. The height of each letter is proportional to its frequency in each set.

  • Fig. 4 PhoQ sequence space.

    (A) Tabulation and examples of double mutants reached by 2, 1, or 0 direct paths from AVST. Functional variants are circled. Lines connect variants differing by one residue (black, accessible paths; gray, inaccessible paths). (B) Cumulative fraction of functional variants reached from wild-type PhoQ in a given number of amino acid (blue) or nucleotide (green) substitutions. (C and D) Examples of shortest paths connecting AVST (wild type) to MLAI and SIQC. Green text indicates residues not in either terminal node. (E and F) Force-directed graphs of functional PhoQ variants (nodes) with edges connecting variants differing by one residue (E) or one nucleotide (F). Node size is proportional to number of neighbors. In (E), clusters are colored with corresponding frequency logos shown. In (F), the color scale represents the probability of reaching a node after 20 mutational steps, with red nodes indicating variants found in PhoQ orthologs. (G) Shortest paths connecting AVST and MLAI via nucleotide substitutions. (H) Frequency logo for interfacial residues of PhoQ orthologs from γ-proteobacteria.

Supplementary Materials

  • Pervasive degeneracy and epistasis in a protein-protein interface

    Anna I. Podgornaia, Michael T. Laub

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Table S2
    • Figs. S1 to S10
    • References
    Table S1
    List of 1,659 sequences identified as functional in our two-step library screen.

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