Superposition of complexes of KPC-2 and CTX-M-15 bound with avibactam.

<p>Superimposed are CTX-M-15 (grey carbons atoms) and KPC-2 (cyan carbon atoms). The deacylation water, W#1, is labelled. Key distance differences listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136813#pone.0136813.t002" target="_blank">Table 2</a> are indicated by dashed lines. These distance differences involve the avibactam N6 atom pointing towards the avibactam C7 atom in CTX-M-15 (listed in black) whereas in KPC-2 this avibactam N6 atom is pointing away thus being situated at a larger distance (listed in green). In addition, the avibactam N6 hydrogen bond with S130 in CTX-M-1 is depicted as well; this is not present in KPC-2.</p

Data collection and refinement statistics for SHV-1 and KPC-2 soaked with avibactam.

<p>Data collection and refinement statistics for SHV-1 and KPC-2 soaked with avibactam.</p

Electron density of avibactam bound to the active site of SHV-1.

<p>|Fo|-|Fc| electron density difference density is depicted for the ligand (contoured at 3.25σ). The covalently-bound avibactam is shown in blue stick model. The map was calculated similar to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136813#pone.0136813.g002" target="_blank">Fig 2</a>.</p

Superposition of complexes of KPC-2 and SHV-1 bound with avibactam.

<p>Superimposed are KPC-2 (cyan carbon atoms) and SHV-1 (grey carbon atoms). The nitrogen N1 is labelled ‘1’ and ‘1’” for KPC-2 and SHV-1, respectively, to indicate the differences in the chirality and direction of the lone pair electrons that nitrogen in the 2 different structures. The oxygen of the sulfate moiety of avibactam bound to KPC-2 is indicated by a ‘*’. The deacylation water, W#1, and sulfate hydrogen-bonding water in KPC-2, W#2, are indicated as well. The following active site residues were used for the superpositioning: SHV-1 residues 233–238, 68–84, 121–140, 167–172 onto the equivalent residues of KPC-2; root-mean-square-deviation is 0.51Å for 48 Cα atoms.</p

Electron density of avibactam bound to the active site of KPC-2.

<p>A, |Fo|-|Fc| electron density difference density is depicted for the ligand (contoured at 3.25σ). The covalently-bound avibactam is shown in blue stick model. Prior to the map calculation, the model with the avibactam ligand removed has been subjected to 10 cycles of Refmac crystallographic refinement. B, close-up view of the electron density around the N1 atom of avibactam with 3 different models of avibactam: avibactam refined with the N1 in the <i>S</i> enantiomer conformation (same colors as in A), avibactam refined with the N1 in the planar conformation (all atoms in magenta), and avibactam refined with the N1 in the <i>R</i> enantiomer conformation (all atoms in green). By changing the refined chirality of the N1 atom, the resulting model yields a different position for the C7 atom of avibactam thus distorting the planarity of the adjacent carbonyl moiety (involving avibactam atoms N1, C7, O, and S70 atom OG). As a measure of this carbonyl planarity, the OG atom distance from the plane defined by avibactam atoms N1, C7, and O is 0.15, 0.56, and 0.86 Å for the <i>S</i>, planar, and <i>R</i> enantiomer conformation of N1, respectively.</p

Interactions of avibactam in the active site of KPC-2.

<p>Avibactam is shown with green carbon atoms. Hydrogen bonds are depicted as dashed lines (cut-off distance is 3.2Å). The deacylation water is present (labeled W#1). Additional waters are labeled W#2–3.</p

Avibactam in the active site of SHV-1.

<p>Interactions of avibactam (shown with green carbon atoms) in the active site of SHV-1. Hydrogen bonds are depicted as dashed lines. The deacylation water is present (labeled W#1). Additional waters are labeled W#2–4.</p

Schematic diagram of avibactam inhibition and deacylation pathways of KPC-2.

<p>Schematic diagram of avibactam inhibition and deacylation pathways of KPC-2.</p

Structure comparison of Cj0843, SLT70, and SLTB3.

<p>The proteins are depicted with a transparent surface and cartoon representation. Cj0843 is shown with the same coloring scheme as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.g001" target="_blank">Fig 1</a>. <i>E</i>. <i>coli</i> SLT70 (PDB ID: 1QTE; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.ref024" target="_blank">24</a>]) 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 <i>P</i>. <i>aeruginosa</i> SLTB3 (PDB ID: 5A07)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.ref027" target="_blank">27</a>] 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.</p

MD snapshots of the 5 disaccharide unit PG strand in the active site of Cj0843 as part of the 1μs simulation.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.g006" target="_blank">Fig 6A</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.g006" target="_blank">Fig 6</a>. The view is slabbed showing a side view that is roughly in a similar orientation as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.g001" target="_blank">Fig 1B</a>. 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 <i>N</i>-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.</p