Comparison of interactions of catalytic aspartates in the structures at pH 2.0 and pH7.0.

<p>Comparison of interactions of catalytic aspartates in the structures at pH 2.0 and pH7.0.</p

Comparison of conformation of catalytic aspartates in the structures at pH 2.0 and pH7.0.

<p>Comparison of conformation of catalytic aspartates in the structures at pH 2.0 and pH7.0.</p

Structural comparison of present complex with tetrahedral intermediate complex [21] and product peptide complex [22]:

<p>Stereo diagram showing the ligand atoms at the catalytic centre along with catalytic aspartates. Protein Cα atoms are used in the structural superposition. WAT1 is within 1 Å from an oxygen atom in the newly generated gem-diol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007860#pone.0007860-Kumar1" target="_blank">[21]</a> or carboxyl group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007860#pone.0007860-Das1" target="_blank">[22]</a>.</p

Fit of carboxy terminal product peptide and active site water molecules into 2Fo-Fc electron density.

<p>The electron density map is contoured at 1.0σ level. The carboxyl product peptide ( violetpurple) and the active site water molecules are shown in the two orientations.</p

Data collection and refinement statistics.

<p>*Data for highest resolution shell are given in the parenthesis.</p

Relative positions of WAT1 and the modelled substrate in the active site:

<p>Diagram showing superposition of three structures: 1) present structure (yellow carbon), 2) unliganded HIV-1 protease (magenta carbon, PDB Id 1LV1) and 3) inactive HIV-1 protease/substrate complex (green carbon, PDB Id 1KJH). Water molecule observed in unliganded HIV-1 protease is also shown (magenta). The distances to the scissile carbon atom are indicated. SA OMIT density contoured at 3σ level is also shown for WAT1.</p

Analysis of SynGUN4 by NMR

<div><p>(A) Comparison of spectra obtained from ¹H-¹⁵N TROSY experiments of SynGUN4 in the absence (black) and presence (red) of 2 mM deuteroporphyrin.</p> <p>(B) Normalized chemical shifts for those ¹H-¹⁵N cross peaks whose positions change in the presence of 2 mM deuteroporphyrin. In general, the largest shifts cluster for residues on the α6/α7 loop. The remaining positions with significant chemical shifts reside on the “greasy palm” region of SynGUN4.</p> <p>(C) Rendered ribbon diagram of the Gun4 core domain with the position of the shifting ¹H-¹⁵N cross peaks mapped onto the backbone structure of SynGUN4. The magnitude of the chemical shift changes shown corresponds to the color bar at the bottom. Briefly, shifts larger than 2.5 parts per million (ppm) are shown in red, shifts between 2 and 2.5 ppm are shown in orange, shifts between 1.5 and 2 ppm are shown in yellow, and shifts of 1.5 ppm and less are shown in green.</p></div

Close-Up View of the GUN4 Core Domain's “Cupped Hand” Architecture

<div><p>(A) Rendered skeletal view of the GUN4 core domain. Helices are shown as red cylinders, and coiled regions are depicted as gray loops. The overall shape resembles that of a “cupped hand.”</p> <p>(B) Rendered view of the solvent-accessible surface of the GUN4 core domain, colored gold. The α6/α7 loop is colored gray and is bound by the remainder of the domain. The “cupped hand” grips this loop.</p></div

Model of a Putative SynGUN4 Porphyrin Complex Compared to an Experimentally Determined Structure for Ferrochelatase Bound to NMMP

<div><p>(A) Comparison of the crystal structure of the B. subtilis ferrochelatase bound to NMMP to the model of the SynGUN4 core domain bound to Mg-Proto. The SynGUN4 core domain • Mg-Proto model was generated by GOLD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030151#pbio-0030151-b54" target="_blank">54</a>]. The carboxylic acid moieties of the porphyrin were staggered between the δ-guanido side chains of Arg214 and Arg217. The position of the arginine loop used to tether the carboxyl moieties of the porphyrin bound to ferrochelatase served as the fixed point for the structural alignment of SynGUN4 and ferrochelatase.</p> <p>(B) Close-up view of the structural alignment between Mg-Proto (gold) and NMMP (lavender). Attempts to strictly superimpose all of the atoms of the two porphyrins resulted in at least one corner of the porphyrin scaffold residing out of the plane defined by the flat Mg-Proto complex, because of the pucker of NMMP.</p></div

Sequence Alignment of GUN4 and GUN4-like Proteins

<div><p>(A) Alignment of the N-terminal portions of GUN4 family members whose N-terminal domains show sequence homology to SynGUN4. Residues contributing to the hydrophobic core of the five-helix bundle are highlighted (pink). GUN4 sequences isolated from plants thus far all have a plastid transit peptide in place of the N-terminal domain found in SynGUN4. The Chlamydomonas reinhardtii sequence was derived from sequence data produced by the United States Department of Energy Joint Genome Institute (<a href="http://www.jgi.doe.gov/" target="_blank">http://www.jgi.doe.gov/</a>). The N-terminal sequence of C. reinhardtii is not yet known but it most likely contains a chloroplast transit peptide.</p> <p>(B) Sequence alignment of possible GUN4 core domains. Residues that form the “palm” of the “cupped hand” are highlighted in pink. Residues from the α6/α7 loop that structure the loop and protrude into the core are highlighted in yellow. Arg214 and Arg217, predicted to be important for binding to porphyrins, are highlighted in blue. Residues that disrupt proper folding when mutated and expressed in E. coli are denoted by an asterisk (*).</p></div