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

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

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

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

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