Structure of MglA and comparsion to SspA and GST proteins.

<p>A) Homodimeric MglA shown from two angles. The right subunit of the dimer is labelled and colored according to its β strands and α helices. B) Superimposition of the MglA dimer (cyan) onto the dimer of <i>Y</i>. <i>pestis</i> SspA (red). C) Superimposition of the MglA dimer (cyan) onto the dimer of nematode <i>C</i>. <i>elegans</i> specific GST (yellow). Although unexpected, MglA forms a dimer that is structurally similar in its arrangement to SspA dimers.</p

Structural and Biochemical Characterization of the <i>Francisella tularensis</i> Pathogenicity Regulator, Macrophage Locus Protein A (MglA)

<div><p><i>Francisella tularensis</i> is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase (Kb) pathogenicity island (FPI) that is regulated by the macrophage locus protein A (MglA) and the stringent starvation protein A (SspA). These proteins interact with both RNA polymerase (RNAP) and the pathogenicity island gene regulator (PigR) to activate FPI transcription. However, the molecular mechanisms involved are not well understood. Indeed, while most bacterial SspA proteins function as homodimers to activate transcription, <i>F</i>. <i>tularensis</i> SspA forms a heterodimer with the MglA protein, which is unique to <i>F</i>. <i>tularensis</i>. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family. Unexpectedly, MglA also formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC) studies showed that MglA is able to self-associate in solution to form a dimer but that it preferentially heterodimerizes with SspA. Finally, the MglA structure revealed malate, which was used in crystallization, bound in an open pocket formed by the dimer, suggesting the possibility that this cleft could function in small molecule ligand binding. The location of this binding region relative to recently mapped PigR and RNAP interacting sites suggest possible roles for small molecule binding in MglA and SspA•MglA function.</p></div

Summary of MglA data collection, processing and refinement statistics.

<p>^a. Values within parentheses refer to the highest shell.</p><p>^b. R_sym = ∑∑|Ihkl—Ihkl(j) |/∑Ihkl, where Ihkl(j) is observed intensity and Ihkl is the final average value of intensity.</p><p>^c. Protein geometry analysis performed by MolProbity⁵⁶.</p><p>^d. R_work = ∑||Fobs| - |Fcalc||/∑|Fobs| and Rfree = ∑||Fobs| - |Fcalc||/∑|Fobs|, where all reflections belong to a test set of 10% data randomly selected in Phenix.</p><p>Summary of MglA data collection, processing and refinement statistics.</p

GST activity measurement of <i>Francisella tularensis</i> MglA, SspA•MglA and pET41a GST.

<p>GST activity measurement of <i>Francisella tularensis</i> MglA, SspA•MglA and pET41a GST.</p

Model of the SspA•MglA heterodimer and visualization of its RNAP and PigR interacting surfaces relative to malate binding sites.

<p>(Left) The surface representation of the Phyre2 model of the SspA•MglA heterodimer with the residues involved in RNAP and PigR binding colored green and tan, respectively. Shown as spheres are the bound malates. Note, the malates bind in the same pocket as PigR. (Right) Views into the faces of the RNAP (top) and PigR/malate (bottom) binding pockets.</p

The MglA-malate binding pockets.

<p>A) Two malate molecules (shown as spheres) are bound in the large cleft of the open face of the MglA dimer. Also shown, as yellow sticks, are the corresponding binding sites of GSH molecules obtained by superimposition of the <i>E</i>. <i>coli</i> GST-GSH complex onto the MglA structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128225#pone.0128225.ref042" target="_blank">42</a>]. The inset below is a simulated annealing omit 2F_o-F_c map (blue mesh), contoured at 1.0 σ and calculated after removal of the malate molecules followed by ten rounds of refinement using phenix.refine, with an initial temperature of 2500 K and a final temperature of 300 K. The map is contoured around the malate molecules. B) Electrostatic surface representation (where blue and red represent positive and negative surfaces, respectively) of the MglA dimer with bound malates (shown as sphere) included to underscore the electropositive nature of their binding region. C) Comparison of binding site of Mal1 (cyan sticks) in MglA with the GSH binding site of two GST proteins. For these analyses, the GST fold of MglA was aligned with those of the <i>E</i>. <i>coli</i> GST (yellow sticks) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128225#pone.0128225.ref042" target="_blank">42</a>] and the <i>M</i>. <i>musculus</i> maleylacetoacetate isomerase (pink sticks) (PDB ID: 2CZ2). D) Superimposition of an MglA subunit onto that of the <i>N</i>. <i>gonorrhoeae</i> GST protein bound to GSH (PDB ID: 4HOJ). The subunits superimpose with an rmsd of 1.9 Å for 181 corresponding Cα atoms. The overlay shows that the catalytic cysteine of the GST protein is replaced by an aspartic acid in MglA, which clashes with the GSH as does the side chain of Tyr11. Moreover, a key GSH interacting loop and residues Lys37, Gln49, Glu62 and Gln106 are absent in the MglA structure.</p

Selected Crystallographic Data and Statistics.

a<p>R_sym = ∑∑|Ihkl-Ihkl(j)|/∑Ihkl, where Ihkl(j) is the observed intensity and Ihkl is the final average value of intensity.</p>b<p>R_work = ∑||F_obs|−|F_calc||/∑|F_obs| and R_free = ∑||F_obs|−|F_calc||/∑|F_obs|; where all reflections belong to a test set of 5% randomly selected data.</p

Structure of reduced C10S Spx in complex with αCTD.

<p>(A) Spx and αCTD are shown as teal and green ribbons, respectively, and their secondary structures are labelled. Helix α4, which is observed in oxidized Spx <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone.0008664-Newberry1" target="_blank">[9]</a> but has unraveled in the reduced form, is colored magenta. The residues mutated in this study, R60 and K62, are labelled and shown as sticks with carbon atoms colored white and nitrogen atoms either blue or magenta. Residues S10 and C13 are labelled and shown as sticks with carbon and sulphur atoms colored yellow and the γ-oxygen of S10, red. (B) Close up of the region surrounding helix α4 and residues C10/S10 and C13 after the superposition of the oxidized and reduced αCTD-Spx complex structures. Reduced Spx is shown as a magenta ribbon and oxidized Spx as a teal ribbon. The C10-C13 disulfide bond is shown in orange sticks and S10 and C13 from the reduced structure are shown as yellow sticks. In the reduced form residue R92 has moved 2.8 Å away from its position in the ammonium sulphate-containing oxidized form <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone.0008664-Newberry1" target="_blank">[9]</a>. The side chain of residue R60 beyond the Cβ atom is disordered in the sulphate-containing crystal form of oxidized Spx, which was used in the superposition visualized here <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone.0008664-Newberry1" target="_blank">[9]</a>.</p

<i>B. subtilis</i> strains and plasmids.

<p>Unless otherwise noted, <i>trxB-lacZ</i> contains a region between -115 and +47 of the <i>trxB</i> promoter.</p

Interaction of αCTD and Spx variants with the regulatory region of the <i>trxB</i> promoter.

<p>The <i>trxB</i> probe (−56 to −21) was generated by annealing of oligonucleotides followed by labeling of the 3′-end of the template strand using Klenow fragment and [³²P]dATP. Bands corresponding to the <i>trxB</i>/αCTD and <i>trxB</i>/Spx/αCTD complexes are marked with arrows. (A) EMSA analysis of αCTD and Spx binding to the <i>trxB</i> probe in reactions containing Spx variant or mixtures of mutant Spx proteins or mutant with the wild-type Spx (each at 5 µM). Abbreviations: W, wild-type Spx; G, Spx^G52R; R, Spx^R60E. (B) Redox-sensitive interaction was examined in the presence of DTT.</p