Figure 5. Structural characterization of multiple constraints on the DHFR mutational landscape.

(A) Mutational response categories from –Lon selection (top, categories in Figure 2C,D) and +Lon selection (bottom, categories as in Figure 2C,D) colored onto residues and displayed on slices as in Figure 1E. (B) Relationship between mutational response and distance from hydride transfer for –Lon selection. The percent of positions from each mutational response category are plotted as a function of distance from the site of hydride transfer. Each category colored as in A), top). (C) Relationship between mutational response and distance from hydride transfer for +Lon selection. Each category colored as in A), bottom).

Figure 5—figure supplement 1. Selection coefficients under the two Lon expression regimes mapped on the DHFR structure.

Structural model of DHFR (PDB ID: 3QL3) in ribbon representation with the DHF substrate and the NADPH cofactor represented by spheres (yellow carbon and heteroatom coloring). The residues are colored in (A,B) by mutational response category from Figure 2C,D for –Lon selection, in (C,D) by mutational response category from +Lon selection, or in (E,F) by the per-position mean ∆selection coefficient from Figure 3.

Figure 5—figure supplement 2. Burial of residues within each mutation response category reported as the mean number of atomic neighbors.

Each point represents one amino acid side chain, and the y-axis reports the average number of heavy atom neighbors within an 8 Å shell for all heavy atoms in that side chain. Box plots are overlaid on the distribution to show the median (orange bar) and upper/lower quartiles. Mutational response categories are shown for both –Lon and +Lon selection. The green arrow highlights the absence of buried Beneficial positions in +Lon selection.

Figure 5—figure supplement 3. Residues in mutational response categories in the –Lon selection as a function of distance from several sites in the DHFR structure.

(A) Location of hydride transfer site, the M20 residue on the M20 loop (orange), and hot spot sites from Figure 4 (the core of the globular domain represented by I41, the beta-sheet surface below the active site represented by L112, and the adenine ring on NADPH) indicated on the DHFR structure (PDB ID: 3QL3). (B–F) The distance relationships between each site and the residues in each mutational response category in the –Lon selection are shown (left) as boxplots with points representing the individual mutants and (right) as curves showing the percent of sequence positions in each mutational response category as a function of distance from the site. Boxplots and curves are colored by mutational response categories from –Lon selection as in Figure 5.