Figure 6. Screening of suppressors for the parent inactive mutant I67V DgkA.

I67 (ball and stick in black) and inter-helical residues in contact with it in either (A) crystal (PDB id 3ZE5 [Li et al., 2013]) or (B) NMR (PDB id 2KDC [Van Horn et al., 2009]) structures of the homotrimeric DgkA are shown. Each monomer of the trimer is shown in a different color. The residues in proximity to I67 in a specific structure are shown in green, while the ones in contact in the alternate structure are highlighted in red. Differential contact pairs for other parent inactive mutants of DgkA have been shown in Figure 6—figure supplement 1. The figure has been prepared using the UCSF Chimera package (developed by Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco [Pettersen et al., 2004]). Second-site suppressor libraries of the parent inactive mutant were constructed by randomizing each of the residue partners present in either of the two structures of the protein. Active mutants were screened on selective media at decreasing NaCl concentrations in an E. coli strain knocked out for dgkA. (C) and (D) show the phenotype of the putative suppressors isolated from the libraries at (C) 0.15% NaCl and 0.01% arabinose and (D) 0% NaCl and 0.01% arabinose. Phenotypes of suppressors corresponding to other parent inactive mutants have been shown in Figure 6—figure supplement 2. (E) A representative plate showing the location of the variants and the controls. '(X)' and '(N)' indicate the structure from which the residue partner has been chosen that is either X-ray or NMR structure, respectively. Parent inactive mutant I67V, WT DgkA and the empty vector pBAD-33 act as reference, positive and negative controls, respectively. The true suppressors are anticipated to grow on the plate with low salt concentration while the parent inactive mutant and (Parent inactive mutant, non-suppressor) pairs fail to grow. Suppressors (I67V, I103L) and (I67V, A104T), which are in spatial proximity in the crystal structure restore the growth defect of the parent inactive mutant I67V, whereas the (I67V, E34L) pair which is in proximity only in the NMR structure, fails to restore the growth defect.

DOI: http://dx.doi.org/10.7554/eLife.09532.017

Figure 6—figure supplement 1. Differential contact residue pairs mapped onto the structures of DgkA.

Parent inactive mutant positions V62 (A, B), M66 (C, D), V68 (E, F) and W112 (G, H) (shown in ball and stick in black) and their corresponding inter-helical contacting partners in the crystal and NMR structures have been mapped onto the (A, C, E, G) X-ray (PDB id 3ZE5 [Li et al., 2013]) and (B, D, F, H) NMR (PDB id 2KDC [Van Horn et al., 2009]) structures of the homotrimeric DgkA protein (shown in ribbon with the three monomers in three different colors). The residues in proximity of the parent inactive mutants in a specific structure are shown in green, while the ones in contact in the alternate structure are highlighted in red. The figure has been prepared using the UCSF Chimera package (developed by Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco [Pettersen et al., 2004]).

DOI: http://dx.doi.org/10.7554/eLife.09532.018

Figure 6—figure supplement 2. Screening for suppressors of parent inactive mutants of DgkA.

Second-site suppressor libraries of the parent inactive mutants V62Q, M66L, M66S and V68G were constructed by individually randomizing each of the partners in both the NMR and X-ray structures of the protein. Suppressors were identified by screening on selective media at decreasing NaCl concentrations in an E. coli strain deleted for dgkA. (A,B), (D,E), (G,H) and (J,K) show the phenotype of the suppressors isolated from the libraries of V62Q, M66L, M66S and V68G, respectively at (A, D, G, J) 0.15% NaCl, 0.01% arabinose, (B,K) 0%NaCl, 0.01% arabinose and (E, H) 0.03% NaCl, 0.01% arabinose. (C, F, I, L) Representative plate photograph showing the location of the variants and the controls for the screening of suppressors of the parent inactive mutants V62Q (A,B), M66L (D,E), M66S (G,H) and V68G (J,K), respectively. '(X)' and '(N)' indicate the structure from which the residue partner has been chosen that is either X-ray or NMR structure, respectively. Parent inactive mutant, Wild-Type (WT) DgkA and the empty vector pBAD-33 act as reference, positive and negative controls, respectively. The true suppressors are anticipated to appear before the parent inactive mutant on the plate when grown in low salt conditions. Suppressors (V62Q, A41G), (M66L, V38A), (M66S, G35A), (V68G, A100V), which are in spatial proximity in the crystal structure restore the growth defect of the parent inactive mutants. A similar analysis for the parent inactive mutant I67V has been shown in Figure 6C–E. Overall, suppressors were only found at positions proximal to the parent inactive mutant in the X-ray structure of DgkA.

DOI: http://dx.doi.org/10.7554/eLife.09532.019