Volume 24, Issue 8, Pages 1237-1247 (August 2016) Role of the PFXFATG[G/Y] Motif in the Activation of SdrG, a Response Regulator Involved in the Alphaproteobacterial General Stress Response  Sébastien Campagne, Sebastian Dintner, Lisa Gottschlich, Maxence Thibault, Miriam Bortfeld-Miller, Andreas Kaczmarczyk, Anne Francez-Charlot, Frédéric H.-T. Allain, Julia A. Vorholt  Structure  Volume 24, Issue 8, Pages 1237-1247 (August 2016) DOI: 10.1016/j.str.2016.05.015 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Illustration of the Y-T Coupling Mechanism On the left side, the inactive conformation of the response regulator is illustrated by a schematic representation. Secondary structure names and conserved amino acids are indicated. The crystal structure of Escherichia coli CheY in its inactive form is also depicted as a ribbon model (PDB: 1JBE; Simonovic and Volz, 2001). On the right side, the active conformation of response regulator is illustrated by a schematic representation on which secondary structure names and important amino acids are labeled. The crystal structure of the active conformation of E. coli BeF3−-CheY (PDB: 1FQW; Lee et al., 2001) is shown as a ribbon representation. This figure illustrates the allosteric transition coined Y-T coupling. The red dashed lines illustrate polar contacts between the BeF3− group and the protein. Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 NMR Study of the SdrG Activation (A) Superimposition of the 15N-1H HSQC fingerprints of Sphingomonas melonis Fr1 SdrG (black spectrum), in the presence of 10 mM MgCl2 (red spectrum) or in its BeF3−-activated state (blue spectrum). (B) Chemical shift perturbation histogram of backbone amide groups observed upon addition of 10 mM MgCl2. (C) Chemical shift perturbation histogram of the backbone amide groups of SdrG observed upon activation (from the inactive to the activated state). At the top of both histograms, the secondary structures of the protein are schematically illustrated. Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Solution Structures of Both Native and Active SdrG (A) Primary sequence of SdrG, the secondary structures are highlighted by blue and brown squares for β strands and α helices, respectively. The sequence of the β3-α3 loop is colored in green and the sequence of the flexible loop located right after the PFXFATGG motif is shown in pink. This color code is conserved in all the figures in this paper. (B) Stereoview of the solution structure of SdrG. The 20 NMR structures are superimposed (PDB: 5IEB). (C) Stereoview of the solution structure of BeF3−-activated SdrG, the 20 NMR structures are superimposed (PDB: 5IEJ). Structural statistics of both NMR structures are given in Table 1. Additional information about the backbone dynamics on the ps-ns time scale are given in Figure S1. Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Structural Changes upon Activation (A) Ribbon representation of the lowest energy model of the solution structure of SdrG. The names of secondary structures and important amino acid side chains are indicated. The red arrows illustrate the movement of side chains or protein parts upon activation. (B) Ribbon representation of the structure of the activated form of SdrG on which the inorganic material (Mg2+ and BeF3−) was modeled. The red lines illustrate putative hydrogen bond contacts that could stabilize the incoming phosphoryl group. (C) Web logo representation of the sequence conservation around the PFXFATG[G/Y] motif across the FAT GUY response regulator family. See also Figure S2 and Table S1. (D) Close-up view of the PFXFATGG motif in the inactive conformation of SdrG. The T83 side chain points out from the phosphorylation site and F94 stacks on top of F79 and F81. (E) Close-up view of the PFXFATGG motif in the activated state of SdrG. In this conformation, the T83 side chain points toward the phosphorylation site and F94 stacks against the side of F81. Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Molecular Basis for SdrG Activation (A–F) Close-up view of the NMR signal of the amide backbone of the residue leucine 100 upon activation of D56A (A) and the mutants K102A (B), G85Y (C), SdrG (D), T83A (E), and T83S (F). This signal was used to probe the activation of the receiver domain. Full spectra are shown in Figure S3. In (D–F), an additional close-up view of the signals of the T83 and G84 amide backbone groups is shown. The black, red, and blue spectra correspond to SdrG in its inactive conformation, after addition of MgCl2, and after addition of beryllium fluoride, respectively. (G) Phosphotransfer reactions from PakB to SdrG variants. Phosphotransfer reactions contained autophosphorylated His6-MBP-PakB (297–507, PakB ∼ P) and different SdrG variants (as indicated) and were allowed to proceed for 30 s (top panel) or 10 min (bottom panel). Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Functional Characterization of SdrG Mutants (A) Measurement of the β-galactosidase activity of a σEcfG-dependent promoter (PnhaA2) fused to a lacZ gene in a ΔsdrG background complemented by different sdrG variants. SdrG and its derivatives were C-terminally fused to YFP and under control of a cumate-inducible promoter. Gray bars represent β-galactosidase activity before induction and black bars represent β-galactosidase activity after 4 hr of induction with 25 μM cumate. Values are given as mean ± SD of three independent experiments. (B) Analysis of the expression of the YFP-SdrG variants before and after cumate induction and of GroEL by western blot. (C) Complementation of ΔsdrG mutant strain and measurement of the salt sensitivity phenotype. A ΔsdrG mutant strain was complemented with wild-type (WT) construct and sdrG derivatives under control of a cumate-inducible promoter. The sensitivity of the sdrG mutants was compared with the WT strain of S. melonis Fr1 and the ΔsdrG mutant strain. These strains harbored the empty plasmid as a control. Dilutions series were spotted on either nutrient broth (NB) or NB supplemented with 300 mM NaCl. For space reasons the strains were spotted onto two separate plates per treatment. Pictures were taken after 3 days of incubation at 28°C. Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Comparison of Sma0114 and SdrG Allosteric Transitions (A) Schematic and ribbon representations of the crystal structure of the inactive conformation of Sma0114 (PDB: 2LPM; Sheftic et al., 2012). (B) Schematic and ribbon representations of the crystal structure of the active conformation of Sma0114 (PDB: 2M98; Sheftic et al., 2014). (C) Schematic and ribbon representations of the crystal structure of the inactive conformation of SdrG (PDB: 5IEB). (D) Schematic and ribbon representations of the crystal structure of the active conformation of SdrG (PDB: 5IEJ). On each scheme and structure, secondary structure names and important side chains are indicated. Red arrows indicate the major structural changes observed upon activation. The red dashed lines illustrate polar contacts between the BeF3− group and the protein in (B) and (D). Structure 2016 24, 1237-1247DOI: (10.1016/j.str.2016.05.015) Copyright © 2016 Elsevier Ltd Terms and Conditions