Volume 24, Issue 8, Pages 1311-1321 (August 2016) Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression  Madhanagopal Anandapadamanaban, Robert Pilstål, Cecilia Andresen, Jill Trewhella, Martin Moche, Björn Wallner, Maria Sunnerhagen  Structure  Volume 24, Issue 8, Pages 1311-1321 (August 2016) DOI: 10.1016/j.str.2016.06.008 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1311-1321DOI: (10.1016/j.str.2016.06.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Crystal Structure of MexR R21W and Pro37-38 Cavity (A) The MexR-R21W dimer structure (gray and blue) featuring Trp21 and Arg73 (labeled), and the DNA recognition helices (H4 and H4′; orange). (B) MexR-R21W and MexR-wt (GH dimer) showing the annotation of (−) and (+) chains, with residue Phe17 highlighted. (C) Graphical plot of asymmetry (André et al., 2008) versus midpoint interdistance of H4-H4′ for MexR (PDB: 1LNW), MexR-R21W (this study), MexR-ox (PDB: 3MEX), MexR-ArmR (PDB: 3ECH), and DNA-bound MepR (black; PDB: 4LLN) and OhrR (gray; PDB: 1Z9C). Distances were measured between Cαs of Arg73/73′ in MexR and between corresponding atoms in MepR and OhrR dimers. (D) MexR-R21W structure highlighting H2/2′, H4/4′, and residues Pro37, Pro38, and Trp21 in both chains. Inset (tilted +45°) shows the 2FO-FC map (gray mesh) of Trp21 and Pro37-38 residues in both chains contoured at 1.2σ. Colors as in (A). (E) Magnified view of Pro37-38 region of MexR-wt (CD, EF, and GH; shown in lines with colors as in C superimposed onto MexR-R21W [shown as sticks, color and orientation as in D]). The adjacent inset similarly shows the comparison with MexR-oxidized and MexR-ArmR. Colors as in (C). (F) Structural representatives for the five largest clusters from the simulation trajectories, superimposed on the dimerization region. (G and H) The Pro37-38 distance distribution (G) and the DNA-binding interhelix orientation distribution (H), taken over the simulation trajectories starting from MexR CD, EF, GH, and R21W dimers. Colors as in (C). See also Figures S1–S4. Structure 2016 24, 1311-1321DOI: (10.1016/j.str.2016.06.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Essential Dynamics and Conformational Spread (A) Essential dynamics analysis done separately on wild-type (top, WT) and mutant (bottom, R21W) simulations. The first principal component (PC1) is shown in the left column and the second (PC2) in the right column for each analysis (WT and R21W), both displayed with two representations. Arrows on structure show the principal direction for each residue of significant perturbation, and stylized arrows show the perceived concerted movements of the corresponding chains as described by the respective principal component. (B) Contour densities of projections of all simulations combined, accounting for 65% of the structural variation. The summed distribution of all trajectories are plotted as gray with subsets CD, EF, GH, and R21W as colored contours on top, normalized with respect to the total (gray) density. (C) Distribution of conformations with less (red, top) and more (red, bottom) than 3.5 Å RMSD to DNA-binding reference structure. Pie charts show how often trajectories from each starting conformation visit each RMSD bin projection; ordered as contour plot. (D) Cluster population of PC1/PC2 projections for the five largest clusters (red) with projections of all conformations as background (gray). Pie charts indicate how often simulations from all starting structures visited each of the five largest clusters. See also Figures S3–S5. Structure 2016 24, 1311-1321DOI: (10.1016/j.str.2016.06.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Protein Structure Network Clusters Calculated from Simulated Residue Side-Chain Interactions and Crystal Observations of Wild-Type and Mutant MexR (A and B) Wild-type (A) and mutant (B) clusters of residues that are in contact throughout most of the simulation are shown as connected spheres, with thickness of connections signifying relative interaction propensity. To enable inspection of individual clusters, these are colored separately and displayed in circles as indicated on the central views; the blue, white, or striped perimeter color indicates whether the clusters predominantly belong to the (−), (+), or both chain conformations. The most significant differences between wild-type and mutant MexR is observed in the size and shape of the clusters connecting the dimerization and DNA-binding domains. (C) Crystal structure observations; wild-type (PDB: 1LNW) open (EF) conformations with arginine stacking highlighted, mutant (PDB: 4ZZL) in closed conformations highlighting Asp29′ and Arg63 interaction, oxidized (PDB: 3MEX) showing cysteine binding in closed conformation. (D) Area of magnification in (C) on the MexR structure. Chain coloring (blue and gray) respectively corresponds to (+) and (−) conformations. See also Table S2 and Figure S6. Structure 2016 24, 1311-1321DOI: (10.1016/j.str.2016.06.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Solution Analysis of MexR-wt and MexR-R21W Using SAXS (A) Log I(q) versus q for both MexR-wt (black squares) and MexR-R21W (red circles) scattering experimental data are shown together with the theoretical curves calculated form their crystal structure. Guinier plots are linear for qRg < 1.3 as expected for monodisperse samples, and the molecular weight calculations correspond to dimer (see Table S1). (B) Overlaid pair-distributions, P(r) profiles for MexR-wt and MexR-R21W. See Experimental Procedures for full description. Structure 2016 24, 1311-1321DOI: (10.1016/j.str.2016.06.008) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Ensemble-Based Model for Ligand- or Mutation-Induced Conformational Restriction In this schematic outline, the MarR family fold is represented by rectangular dimerization domains and circular DNA-binding domains. The wide range of accessible states in the free ensemble is indicated by multiple circles representing the DNA-binding domains, where one of these states (light orange) is capable of DNA binding. For simplicity, the common property of one or more ligand-binding cavities in MarR family proteins is represented by a single empty sphere. Based on our findings, we propose that ligand binding or mutations select and stabilize states that are not compatible with DNA binding but are already present in the free ensemble, thereby disabling DNA binding and derepressing the corresponding gene. Structure 2016 24, 1311-1321DOI: (10.1016/j.str.2016.06.008) Copyright © 2016 Elsevier Ltd Terms and Conditions