Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

Characterization of a large family of outer membrane channels from gram-negative bacteria suggest how they can thrive in nutrient-poor environments and how channel inactivation can contribute to antibiotic resistance

Data collection, structure solution, and refinement statistics for Cj0843 data sets.

<p>Data collection, structure solution, and refinement statistics for Cj0843 data sets.</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

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from <i>Campylobacter jejuni</i>

<div><p>The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from <i>Campylobacter jejuni</i> with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of <i>E</i>. <i>coli</i> SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.</p></div

A proposed mechanism of PG hydrolysis by Cj0843.

<p>The colors of the domains of Cj0843 are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.g001" target="_blank">Fig 1</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.s009" target="_blank">S9 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197136#pone.0197136.s015" target="_blank">S3 Video</a>.</p

OccK Channels from <i>Pseudomonas aeruginosa</i> Exhibit Diverse Single-Channel Electrical Signatures but Conserved Anion Selectivity

<i>Pseudomonas aeruginosa</i> is a Gram-negative bacterium that utilizes substrate-specific outer membrane (OM) proteins for the uptake of small, water-soluble nutrients employed in the growth and function of the cell. In this paper, we present for the first time a comprehensive single-channel examination of seven members of the OM carboxylate channel K (OccK) subfamily. Recent biochemical, functional, and structural characterization of the OccK proteins revealed their common features, such as a closely related, monomeric, 18-stranded β-barrel conformation with a kidney-shaped transmembrane pore and the presence of a basic ladder within the channel lumen. Here, we report that the OccK proteins exhibited fairly distinct unitary conductance values, in a much broader range than previously expected, which includes low (∼40–100 pS) and medium (∼100–380 pS) conductance. These proteins showed diverse single-channel dynamics of current gating transitions, revealing one-open substate (OccK3), two-open substate (OccK4–OccK6), and three-open substate (OccK1, OccK2, and OccK7) kinetics with functionally distinct conformations. Interestingly, we discovered that anion selectivity is a conserved trait among the members of the OccK subfamily, confirming the presence of a net pool of positively charged residues within their central constriction. Moreover, these results are in accord with an increased specificity and selectivity of these protein channels for negatively charged, carboxylate-containing substrates. Our findings might ignite future functional examinations and full atomistic computational studies for unraveling a mechanistic understanding of the passage of small molecules across the lumen of substrate-specific, β-barrel OM proteins

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

Bulgecin A binding to Cj0843.

<p>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.</p

The structure of soluble lytic transglycosylase of Cj0843.

<p>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).</p