Penicillin and moenomycin direct mCh-cw to the cross wall, irrespective of SP type.

<p><b>A.</b> Schematic representation of antibiotics treatment assay. Untreated (<b>□</b>); treated with penicillin (0.02 µg/ml) (•); treated with moenomycin (flavomycin) (1 µg/ml) (×). <b>B.</b> Influence of penicillin (Pc) and moenomycin (Fla) on the subcellular localization of mCh-cw hybrid proteins. Arrowheads indicated the cross wall accumulation of mCh-cw; arrows indicated the ring-like distribution; scale bar, 2 µm.</p

Schematic representation of mCh-hybrids.

<p>SP, signal peptide; PP, propeptide; CWS, cell wall sorting signal; mCh: mCherry; lip, lipase. The amino acid sequence of CWS was indicated. The parent plasmid was pCX30 and all mCh-fusion constructs were under control of the xylose-inducible promoter, P<i>xyl</i>.</p

Monitoring mCh-hybrids.

<p><b>A</b>. Fluorescence intensity comparison of mCh-hybrids from different cell fractions. WT-cyto, SA113 (pCXmCh-cyto); WT-cw1 or 2, SA113 (pCXmCh-cw1) or (pCXmCh-cw2); WT-sec1 or 2, SA113 (pCXmCh-sec1) or (pCXmCh-sec2); Δ<i>srtA</i>-cw1 or 2, SA113 Δ<i>srtA</i> (pCXmCh-cw1) or (pCXmCh-cw2); lys, lysostaphin. <b>B.</b> Western blotting of mCh-hybrid proteins in the culture supernatant of protein A deficient mutant SA113 Δ<i>spa</i>. Blank, SA113 Δ<i>spa</i> without plasmid; cyto, SA113 Δ<i>spa</i> (pCXmCh-cyto); cw1 or 2, SA113 Δ<i>spa</i> (pCXmCh-cw1) or (pCXmCh-cw2); sec1 or 2, SA113 Δ<i>spa</i> (pCXmCh-sec1) or (pCXmCh-sec2); Δ<i>srtA</i>-cw1 or 2, SA113 Δ<i>spa</i>Δ<i>srtA</i> (pCXmCh-cw1) or (pCXmCh-cw2). <b>C.</b> Subcellular localization of mCh-hybrid proteins in SA113. <b>a.</b> pCXmCh-cw1; <b>b.</b> pCXmCh-cw2; <b>c.</b> pCXmCh-sec1; <b>d.</b> pCXmCh-sec2; <b>e.</b> pCXmCh-cyto. Arrowheads in <b>a</b> and <b>b</b>, fluorescence localized at the cross wall in <b>a</b>, but absent from the cross wall in <b>b</b>; arrows in <b>a</b> and <b>c,</b> RF foci close to the initial sites of the cross walls; arrowheads in <b>d,</b> halo-like RF distribution absent from the cross wall. Images <b>a, c,</b> and <b>e</b> were taken after one hour of xylose induction; images <b>b</b> and <b>d</b> were taken after two hours of induction. Green: Van-FL staining (cell wall); scale bar, 2 µm.</p

Penicillin and moenomycin treatment led to enrichment of Van-FL at the cross wall.

<p><b>A.</b> Fluorescence intensity profile of Van-FL staining from a line perpendicular to the cross wall and across the middle point of the cross wall. Simple line, untreated cell; dotted line with filled squares, moenomycin (Fla) treated cell; line with filled circles, penicillin (Pc) treated cell. Max amplitude represented the cross wall intensity. Note that the figure was remade using ImageJ software from the microscopy images; the intensity and distance values were not the same as the original data from Leica AF software; but represented the same profile distribution. <b>B.</b> Comparative Van-FL intensity at the cross wall among untreated, penicillin (Pc) treated, and moenomycin (Fla) treated cells. The cross wall Van-FL intensity values were calculated by the ratio of max amplitude against mean fluorescence intensity (generated by Leica AF software) from the same cell. The average ratio of 150 cells from three independent experiments of each group was shown in the bars. White bar, SA113 (pCXmCh-cw1); gray bar, SA113 (pCXmCh-cw2).</p

Localization patterns of Δ<i>srtA</i> (pCXmCh-cw1&2) in the presence of penicillin or moenomycin.

<p>Arrows, mCh-cw dispersed over the entire cell; arrowheads, the cross wall localized mCh-cw. Scale bar, 2 µm.</p

Results of docking of AmiE with the MTP ligand.

<p>(A) Putative interactions between AmiE (grey) and MTP (orange) in the docking model. AmiE residues forming hydrogen bond are colored green, residues making three or more van der Waals interactions are colored blue. Hydrogen bonds are indicated with dashed lines. (B) Surface representation of AmiE with MTP docked into the binding groove. Residues forming hydrogen bonds and van der Waals interactions were calculated using the PISA server (<a href="http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html" target="_blank">http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html</a>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000807#ppat.1000807-Krissinel1" target="_blank">[39]</a>. The putative locations of glycine residues, which would be attached to the lysine side chain in the natural ligand, are indicated with yellow circles. (C) Schematic representation of interactions between AmiE and MTP. Van der Waals interactions are represented with arcs. The same color scheme for contacting residues was used in panels A, B and C. (D) Summary of the observed hydrogen bonds in the model.</p

Crystal structure of the catalytic domain of AmiE.

<p>(A) Cartoon representation of the AmiE crystal structure. Two views differing by 90° are shown. Helices and strands are colored blue and orange, respectively. The zinc ion bound in the active center is shown as a grey sphere. (B) Domain arrangement of the bifunctional AtlE precursor protein. Arrows indicate the post-translational cleavage sites. <b>SP</b> signalpeptide, <b>PP</b> pro-peptide, <b>cat</b> catalytic domains, <b>R1 R2 R3</b> repeat domains. (C) Topology diagram of the AmiE structure, with helices and strands represented with cylinders and arrows, respectively. Numbers of amino acids are given in rectangles.</p

Structural comparison of AmiE with homologous proteins.

<p>(A) Representation of the overall fold. Helices are shown as cylinders, strands as arrows. Zinc ions in the active centers are shown as spheres. R.m.s. deviations and Z-scores were calculated by the DALI server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000807#ppat.1000807-HolmL1" target="_blank">[15]</a>. PlyL (<i>B. anthracis</i> prophage) has the strongest structural homology to AmiE (Z-score = 18.4, rmsd = 2.1). This is followed by <i>D. melanogaster</i> PGRP-LB (Z-score = 12.4, rmsd = 2.3), <i>H. sapiens</i> PGRP-S (Z-score = 12.4, rmsd = 2.4), <i>H. sapiens</i> PGRP-IαC (Z-score = 12.3, rmsd = 2.3) and <i>D. melanogaster</i> PGRP-SA (Z-score = 12.1, rmsd = 2.4). Alignments were calculated with the program lsqkab from the CCP4 suite <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000807#ppat.1000807-Collaborative1" target="_blank">[23]</a>. (B) Close-up view on the superimposed active sites. Zinc-coordinating residues and residues participating in catalysis are labeled and color-coded. (C) Superimposition of structurally conserved asparagine residues making contacts with the second and third amino acid in the peptide stem of MTP.</p

Close-up view of the AmiE active center and mechanism of catalysis.

<p>(A) Architecture of the active site. Side chains of H60, H165, D179 (blue) and a water molecule (red) coordinate a central zinc ion. Side chains of H177 and E119 (green) are 4.5 Å and 4.9 Å, respectively, apart from the zinc. E119 likely acts as a proton shuttle while the protonated side chain of H177 probably serves to stabilize a transition state. (B) Proposed mechanism of catalysis. The free enzyme is shown in (I). Upon docking of a PGN-fragment the Michaelis-Menten complex is formed (II). Acting as an electrophilic catalyst, the zinc ion accepts an electron pair from the carbonyl oxygen of the lactyl moiety, which becomes wedged between the water molecule and the side chain of H177. This results in a pentacoordinated zinc ion and a displacement of the water molecule towards the E119 side chain. The strong polarization between the positively charged zinc ion and the negative carboxylate of E119 leads to a nucleophilic attack of the water oxygen on the carbonyl carbon, which is in close vicinity. In this process, E119 serves as a proton shuttle by transferring the accepted proton to the nitrogen of the peptide bond. This results in the formation of a transition state (III), in which the former carbonyl carbon is now tetrahedral. The negative charge on the carbonyl oxygen in this state is stabilized by the protonated side chain of H177. In the next step (IV), E119 acts again as a proton shuttle by transferring the second proton. Thus, it promotes cleavage of the peptide bond and subsequent release of the peptide stem. In this state, MurNAc is still attached to the zinc ion via the lactyl carboxyl-group. Replacement against an incoming water molecule closes the catalytic cycle and reconstitutes the initial state (I).</p

MTP digestion assay.

<p>ESI-MS spectra of MTP digest with AmiE wt and H177A. The wildtype enzyme and an inactive mutant (H177A) were incubated for 72h with MTP. ESI-MS spectrum of AmiE wt + MTP (left). Arrows indicate the masses of the digestion products. The main peaks are at m/z 346 and 374, which correspond to the cleaved peptide stem. ESI-MS spectrum of MTP + H177A (right). The m/z peaks at 621 and 649 correspond to MTP as synthesized.</p