The non-catalytic domain of PDIR contains a conserved positively charged surface.

<p>(A) Mapping of sequence conservation on the surface of the human PDIR domain; invariant residues are colored green. (B) Surface charge; the positively charged (blue) surface coincides with the conserved region. Negative charge is in red. (C) The conserved lysine and arginine residues are located on helices α1 and α3. The domain orientation in panels (B) and (C) is identical to that in the left view of the panel (A).</p

The PDIR non-catalytic domain binds to the P-domain of calreticulin.

<p>(A) Downfield region of HSQC spectra of ¹⁵N-labeled P-domain (residues 211–261) titrated with increasing amounts of the non-catalytic domain of PDIR. The spectra show specific chemical shift changes for residues Ile225 and Ile249. (B) Plot of weighted-average ¹H and ¹⁵N chemical shift changes in the ¹⁵N-labeled calreticulin P-domain upon addition of the unlabeled PDIR domain. (C) Mapping of the chemical shifts measured onto the NMR structure of the calreticulin P-domain (PDB code 1k9c). Magenta indicates a large chemical shift change (>0.1 ppm); white indicates no change detected. Residues showing chemical shift changes above 0.07 ppm are labeled. (D) Surface charge representation of the P-domain. Negative charge is shown in red, positive charge is in blue. (E) Titration of the ¹⁵N-labeled P-domain with the PDIR non-catalytic domain in the presence of 0.5 M ammonium sulfate. The overlay corresponds to the P-domain/PDIR molar ratio of 1∶0 (red), 1∶1 (yellow) and 1∶2 (blue). (F) Titration of the ¹⁵N-labeled PDIR non-catalytic domain with increasing amounts of unlabeled P-domain results in shifts and disappearance of a number of peaks. Overlay shows spectra at the PDIR/P-domain molar ratio of 1∶0 (red), 1∶1 (yellow), 1∶2 (cyan), 1∶4 (purple) and 1∶8 (blue).</p

Sequence analysis of the PDIR non-catalytic domain.

<p>(A) Occurrence of the domain in protein disulfide isomerases and other proteins. Human ERp57 is shown for comparison. Catalytic motifs are shown in catalytically-active thioredoxin-like domains. (B) Rooted phylogenetic tree of proteins shown in panel (A). Sequences labeled WUBG_02370 and RNA methyltransferase are proteins from parasitic nematodes <i>Wuchereria bancrofti</i> (EJW86719) and <i>Brugia malayi</i> (XP_001896925); mosquito PDIR is from <i>Aedes aegypti</i> (XP_001659136). The N-terminal catalytic domain of ERp57 was used for the phylogenetic tree. The figure was generated with ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062021#pone.0062021-Thompson1" target="_blank">[29]</a> and TreeViewPPC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062021#pone.0062021-Page1" target="_blank">[30]</a>. (C) Sequence alignment of the non-catalytic domain from PDIR proteins from human (NP_006801), rabbit (XP_002716857), rattlesnake (AFJ50881), chicken (XP_422097), zebrafish (XP_001107048), frog (XP_001086600), fly (XP_609645), and sea urchin (XP_001200801) and the related sequence from <i>Brugia malayi</i> RNA methyltransferase (XP_001896925). The consensus sequence is shown below; the secondary structure elements are above the sequence.</p

Data collection and refinement statistics.

1<p>Highest resolution shell is shown in parentheses.</p>2<p>E.S.U.—estimated overall coordinate error based on maximum likelihood.</p>3<p>Stereochemistry was computed using PROCHECK.</p

Crystal contacts identify a putative binding surface for hydrophobic polypeptides.

<p>(A) The Leu143 and Trp144 from the C-terminal tail of the crystallized fragment bind to a pocket in an adjacent PDIR molecule. (B) The base of the pocket is lined with hydrophobic residues, while Glu31 and Arg46 make hydrogen bonds with backbone amide and carbonyl groups.</p

Crystal Structure of the <i>Salmonella</i> Typhimurium Effector GtgE

<div><p><i>Salmonella</i> Typhimurium GtgE is an effector protein contributing to the virulence of this pathogen. It was shown to possess highly selective proteolytic activity against a subset of Rab proteins that helps in evasion of <i>Salmonella</i>-containing vacuole (SCV) fusion with lysosomes. Cys45, His151 and Asp169 are essential for proteolytic activity. The structure of a C-terminal fragment GtgE(79–214) indicated the presence of a papain-like fold. Here, we present the structure of GtgE(17–214) containing the fully assembled active site. The design of a proteolytically active and crystallizable GtgE construct was aided by NMR spectroscopy. The protein indeed displays papain-like fold with an assembled Cys-His-Asp catalytic triad. Like the full-length GtgE, the crystallizable construct showed low activity <i>in vitro</i> for its known substrates, Rab32 and Rab29. NMR titration experiments showed at most very weak binding of GtgE to the peptide encompassing the Rab29 cleavage site. In view of the low <i>in vitro</i> activity and poor substrate binding, we postulate that the function of GtgE <i>in vivo</i> as a proteolytic enzyme is dependent on other factor(s), such as a protein partner or interactions with the SCV membrane, which stimulate(s) GtgE activity <i>in vivo</i>.</p></div

¹H,¹⁵N-correlation HSQC spectra of GtgE.

<p>(A) GtgE(17–214), dispersion of peaks is characteristic of a well-folded protein. (B) Superposition of HSQC spectra of GtgE(17–214) (red) and GtgE(79–214) (cyan).</p

Statistics of data collection and refinement.

<p>Statistics of data collection and refinement.</p

Structure of GtgE(17–214, Δ33–40, C45S).

<p>(A) Cartoon representation with the active site residues shown in a stick representation and marked with one-letter code. Position of Ser45 is marked with letter C to indicate that this residue is a cysteine in the wild type GtgE. The molecule is colored in rainbow, from blue at the N-terminus to red at the C-terminus. The disordered segment is shown by a dashed line. The extended termini contact symmetry-related molecules in the crystal. The strand ß1 extends ß-sheet of the neighboring molecule. The catalytic residues and the four C-terminal histidines are shown in a stick mode. Secondary structure elements are labeled. This and subsequent figures were prepared with PyMol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). (B) The topology diagram of GtgE. The location of catalytic residues is marked with letters C, H and D. (C) The superposition of GtgE (colored rainbow, this work) and the GtgE(79–214) fragment (magenta, PDB code 4MI7, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166643#pone.0166643.ref022" target="_blank">22</a>]). The ~20 swapped N-terminal residues of the symmetry related molecule are depicted here to show the structure of an intact GtgE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166643#pone.0166643.g003" target="_blank">Fig 3C</a>). The N- and C-termini are marked, the active site residues, Ser45 (Cys45 in wild type GtgE), His151 and Asp169 are shown in stick mode and colored white for GtgE and cyan for GtgE fragment. The inserts on the right shows the expanded view of the active site as observed in our structure. A sulfate molecule is bound near the catalytic histidine and forms hydrogen bonds with its ND1 atom as well as with Tyr42 and Arg142 sidechains. In the presence of a substrate the His151 sidechain would flip by 180° to form hydrogen bond between its ND1 nitrogen and SG of Cys45. Green dashed line indicates the hydrogen bond between His151 and Asp169 observed in the GtgE but not in the GtgE(79–214) fragment.</p

Effects of MgCl₂ and EDTA on the interaction between F12-ATP and GST-HEPN.

<p>A. F12-ATP vs. GST-HEPN FP titration was carried out in the presence of 1 mM MgCl₂ and increasing concentrations of EDTA. B. F12-ATP vs. GST-HEPN FP titration with increasing concentrations of EDTA alone. C. EDTA-interacting residues are labeled in <i>blue</i> on the HEPN crystal structure. D. G-tetra-P-binding residues are mapped onto HEPN. G-tetra-P-binding residues that are also involved in GTP-binding are labeled in <i>blue</i> and additional residues only involved in G-tetra-P-binding are labeled in <i>red</i>.</p