Enzyme activity of human carboxylesterases.

<p>Plots of initial rates of reaction against enzyme concentration assayed as described in the Methods section at a substrate concentration of 750 μM 4-NPA (a) CES1 (b) CES1 N79Q. Plots of initial reaction rates against substrate concentration for the hydrolysis of 4-NPA by (c) hCES1 and (d) hCES1 N79Q (3.4 nM each enzyme). The molarity of the enzyme was calculated assuming 100% trimer with a molecular weight of 182.7 kDa.</p

Purification of human carboxylesterases.

<p>(a) Size exclusion profiles of purified hCES1 (blue trace) hCES1 N79Q (green trace) and hCES1 S221A (red trace) enzymes from media of transfected HEK cells. Samples were run on a HiLoad 16/60Superdex 200 column (GE Healthcare) in 200 mM NaCl, 20 mM Tris-HCl, pH 7.5. The peak corresponds to a molecular weight of approximately 160 kDa as estimated from the elution volumes of globular proteins of known molecular weight: Aprotinin (6.5 kDa) Ribonuclease A (13.7 kDa) Carbonic Anhydrase (29 kDa), Ovalbumin (44 kDa), Conalbumin (75 kDa) Aldolase (158 kDa) Ferritin (440 kDa) and Blue Dextran 2000. (b) SDS-polyacrylamide gel of purified CES1 (lanes 1 and 2) and CES1 N79Q (lanes 3 and 4) untreated (lanes 1 and 3) and treated with PNGaseF (lanes 2 and 4). (c) The sedimentation velocity distribution for hCES1 N79Q. Data for hCES1 and hCES1 S221A gave the same profiles.</p

Refinement statistics for native and selenomethionine substituted AmyR.

<p>Refinement statistics for native and selenomethionine substituted AmyR.</p

Data collection statistics for native and selenomethionine substituted AmyR.

<p>Values in parentheses are for the highest resolution shell.</p

Rotated views of the <i>E</i>. <i>amylovora</i> AmyR dimer.

<p>(A) Ribbon diagram, coloured by secondary structure. (B) Surface topography, coloured by surface charge at pH 7.4, red = negative, blue = positive.</p

Ribbon representation of the overall structure of the AmyR monomer.

<p>The AmyR monomer is coloured by secondary structure. Secondary structure elements are labelled with residue numbers. The right panel is rotated 90°. β-strand 1 interacts with the equivalent residues in the dimer, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176049#pone.0176049.g002" target="_blank">Fig 2</a>.</p

Structural alignment of AmyR monomer with other YbjN and T3C proteins.

<p>The two views are rotated horizontally 90°. The alignment includes the PDB entries 1JYO:D (<i>Salmonella enterica</i> SicP, ice blue), 1S28:A (<i>Pseudomonas savastanoi</i> AvrPphF ORF1, gold), 1TTW:A (<i>Yersinia pestis</i> SycH, coral), 1XKP:B (<i>Yersinia pestis</i> SycN, grey), 2PLG:B (<i>Synechococcus elongates</i> T110839, pink), 3EPU:A (<i>Salmonella enterica</i> STM2138, sea green), 3KXY:J (<i>Escherichia coli</i> ExsC, brown), and 4H5B:A (<i>Deinococcus radiodurans</i> DR_1245, lilac), and 5FR7:A (<i>Erwinia amylovora</i> AmyR, yellow).</p

Unmodelled density at crystal contact between molecules.

<p>(A) Electron density and difference map of the unmodelled density at the crystal contact. The main crystal contact forming density is central, while the two other chains are seen below. (B) An alternative view of the two chains.</p

The water molecules between the dimer interface.

<p>Cross-section of the hydrophilic pocket at the dimer interface, showing the A and B chains coloured in gold and silver, with the highly ordered water molecules contained within.</p

The crystal structure of <i>Erwinia amylovora</i> AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions

<div><p>AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species <i>Erwinia amylovora</i>, and is a functionally conserved ortholog of YbjN from <i>Escherichia coli</i>. The crystal structure of <i>E</i>. <i>amylovora</i> AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.</p></div