Fig 1.
The structure of soluble lytic transglycosylase of Cj0843.
A Front view of Cj0843 depicting the NU domain (teal), NU-loop (magenta), U-domain (blue), UL-loop (blue-green), L domain (yellow), and C-domain (red). The disulfide bond between C87 and C102 is in green stick model, and the catalytic E390 is shown in black spheres. B Side view of Cj0843 (90 degrees rotated along a vertical axis relative to the orientation in A).
Fig 2.
Structure comparison of Cj0843, SLT70, and SLTB3.
The proteins are depicted with a transparent surface and cartoon representation. Cj0843 is shown with the same coloring scheme as in Fig 1. E. coli SLT70 (PDB ID: 1QTE; [24]) has a similar coloring scheme except it does not have an NU-domain. The SLT70 structure includes a 1,6-anhydromurotripeptide (black sticks) to highlight the location of the active site; the disulfide bond is shown in green. The P. aeruginosa SLTB3 (PDB ID: 5A07)[27] is depicted with a similar muropeptide ligand shown in black sticks; the N-terminal domain (light blue), catalytic domain (red), PG binding domain (purple), and αβ-domain (pink) are shown.
Fig 3.
A Unbiased |Fo|-|Fc| difference density contoured at 3σ contour level showing the presence of bulgecin A in the active site (bulgecin A was removed from the map calculations). Bulgecin A is depicted with carbon atoms colored in cyan. B Interactions of bulgecin A in the active site; interacting water molecules are shown as red spheres. Hydrogen bonds are depicted as dashed lines. C Active site movements of Cj0843 upon bulgecin A binding. The bulgecin A complex (cyan), apo Cj0843 P3121 structure (red), apo Cj0843 I23 structure (orange) are superimposed to highlight the main chain movements and the F412 side chain movement. The view is roughly 90° rotated from the view in A and depicts the active site groove from the side. Residues M410, Y463, and the catalytic E390 are shown in stick model.
Fig 4.
Enzymatic and microbiological assays of Cj0843 with bulgecin A.
A Turbidity assay shows inhibition of 1μM Cj0843 by bulgecin A (0.25–250μM). Control experiments include adding no enzyme and boiled/inactivated Cj0843. Data show the mean ± SEM of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 (Student’s t-test). B Bulgecin A potentiated the efficacy of Amp against β-lactam resistant C. jejuni. A β-lactam resistant C. jejuni strain was inoculated in MH broth (control), or MH broth supplemented with Amp (128 μg/mL, sublethal concentration), bulgecin A (100 μg/mL) or both Amp (128 μg/mL) and bulgecin A (100 μg/mL). The detection limit (dotted line) of the assay is 1 x 103 CFU/mL. Each data point represents the mean ± SEM obtained from duplicate wells in the microtiter plate growth assay. At 30hr, MH vs Amp: P = 0.01; Amp vs Bul: P = 0.04; Bul vs Amp+Bul: P = 0.0001; Amp vs Amp+Bul: P < 0.0001 (Student’s t-test).
Fig 5.
The electrostatic surface potential of Cj0843, SLT70, and LtgA.
The electrostatic surface calculations were done using APBS [41], and two opposite views of the doughnut-shaped proteins are shown. The active site groove is indicated by an arrow and labeled ‘1’; the positively charged pocket 2 is indicated by a yellow ‘2’.
Fig 6.
Molecular snapshots of PG strands in the Cj0843 active site modeled in substrate and product-binding modes.
A A 5 disaccharide unit PG strand modeled as a substrate in the active site. A dashed red line highlights the terminal disaccharide peptide unit to be cleaved off. The PG tetrapeptide sections are shown with yellow carbon atoms. The PG GlcNAc (‘G’), MurNAc (‘M’), and 1,6-anhydroMurNAc (‘AnhM’) moieties are shown with light blue, blue/green, dark blue carbon atoms, respectively. Arg and Lys residues in the pocket 2 found to interact with the carboxyl moieties of the tetrapeptide are shown with light green carbon atoms. Key active site residues are labeled including the catalytic E390. B A 4 disaccharide unit PG strand modeled as a product in the active site.
Fig 7.
MD snapshot of the 5 disaccharide unit PG strand in the active site of Cj0843 in the substrate-binding mode.
A The snapshot is at time point 57.82 ns of the protonated E390 MD run 3. The coloring and labeling of the PG strand are the same as in Fig 6. Hydrogen bonds are depicted as dashed black lines. MurNAc-1 (‘M-1’) is observed to be in a boat conformation. Hydrogen atoms for PG and protein are not depicted except for the hydrogens on the protonated side chain of E390 and on the O6 atom of MurNAc-1. B Schematic diagram of the PG strand interactions in the active site of Cj0843 (left, from 57.82 ns time point) compared to the crystal structure of bulgecin A bound in the active site (right).
Fig 8.
MD snapshots of the 5 disaccharide unit PG strand in the active site of Cj0843 as part of the 1μs simulation.
A The starting position and starting conformation of the PG strand after minimization but before the NVT. The tetrapeptide moieties of the terminal disaccharide unit of the PG strand are labeled ‘51’ through ‘54’. The GlcNAc and MurNAc moieties are shown with grey carbon atoms whereas the tetrapeptide moieties are shown with orange colored carbon atoms; these PG moieties are labeled in purple. For reference, this and subsequent snapshots also contains a PG strand in the active site as obtained from the simulations with the PG in the substrate-binding mode (same binding mode and coloring as in Fig 6A yet with black italicized labels). The 1,6-anhydroMurNAc moiety is colored darker and labeled bold ‘aM+2’ for both PG strands in each panel. The labels of the moieties of the terminal disaccharide unit to be cleaved off in both the MD PG strand and the reference PG substrate strand are underlined. Residue E390 is labeled and shown in black sticks, and the Arg/Lys residues are shown with green carbon atoms as in Fig 6. The view is slabbed showing a side view that is roughly in a similar orientation as Fig 1B. B The PG strand after the NVT and NPT equilibration step but before the production MD run. C Snapshot at 150ns showing the PG has entered the pore but is bound unproductively distant from the active site E390. D Snapshot at 559.6ns showing that the terminal tetrapeptide section has reached the positively charged pocket 2 and makes similar carboxyl interactions as the equivalent tetrapeptide section in the comparison substrate modeled PG strand (arrows). At this latter time point, the glycan strand has approached the active site the closest within the entire 1μs simulation; the nitrogen of the N-acetyl moiety of GlcNAc-2 residue is within 7Å the Y463 main chain oxygen. Also, the anchoring in pocket 2 lined up the correct MurNAc-1 and GlcNAc+1 with respect to their equivalent moieties in the substrate-binding mode (red dashed lines) to facilitate cleavage of the terminal disaccharide PG unit pending the final approach to the active site groove.
Fig 9.
A proposed mechanism of PG hydrolysis by Cj0843.
The colors of the domains of Cj0843 are as in Fig 1. The 8 R/K residues in pocket 2 are indicated, and several additional R/K labels are drawn for illustrative purposes. In addition to E390 (black sphere), M410 and Y463 are labeled ‘M’ and ‘Y’, respectively. A narrowing of the active site groove is depicted in states 6–8 with an accompanying shift of the flanking NU-domain. The boat conformation of MurNAc-1 is drawn in state 6. The tetrapeptide sections of the 5 PG disaccharide units are colored as in Figures F and K in S9 Fig and S3 Video.
Table 1.
Data collection, structure solution, and refinement statistics for Cj0843 data sets.
Table 2.
Overview of different molecular dynamics simulations carried out for Cj0843 with PG.