The Crystal Structure of OprG from Pseudomonas aeruginosa, a Potential Channel for Transport of Hydrophobic Molecules across the Outer Membrane

Background: The outer membrane (OM) of Gram-negative bacteria provides a barrier to the passage of hydrophobic and hydrophilic compounds into the cell. The OM has embedded proteins that serve important functions in signal transduction and in the transport of molecules into the periplasm. The OmpW family of OM proteins, of which P. aeruginosa OprG is a member, is widespread in Gram-negative bacteria. The biological functions of OprG and other OmpW family members are still unclear. Methodology/Principal Findings: In order to obtain more information about possible functions of OmpW family members we have solved the X-ray crystal structure of P. aeruginosa OprG at 2.4 A ˚ resolution. OprG forms an eightstranded b-barrel with a hydrophobic channel that leads from the extracellular surface to a lateral opening in the barrel wall. The OprG barrel is closed off from the periplasm by interacting polar and charged residues on opposite sides of the barrel wall. Conclusions/Significance: The crystal structure, together with recent biochemical data, suggests that OprG and other OmpW family members form channels that mediate the diffusion of small hydrophobic molecules across the OM by a latera

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

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Structural and spectroscopic studies of heavy metal binding to de novo designed coiled -coil peptides.

Structural characterization of both apo and As(III) bound cysteine-containing designed three-stranded coiled coil peptides and spectroscopic studies to determine Pb(II) and Cd(II) binding specificity to peptides with two thiolate metal binding sites were performed to better understand the interactions of these toxic metals with cysteine rich biomolecules. Crystallographic determination of the structures of the peptides CSL19C [Ac-EWEALEKKLAALESKLQACEKKLEALEHG-NH 2] and As(CSL9C)3 the As(III) complex of CSL9C, [Ac-EWEALEKKCAALESKLQALEKKLEALEHG-NH 2]. UV-Vis, CD, 113Cd NMR, and NOESY 2D NMR spectroscopic techniques used to evaluate metal binding to the disubstituted peptides. The parallel structure obtained for the Coil Ser derivative CSL19C differs from the antiparallel orientation obtained for its parent peptide. The orientation of cysteine side chains in the d position away from the center of the coiled coil experimentally confirms hypotheses from modelling studies and provides a basis on which to understand metal binding to these sites. Additionally, the discovery of Zn(II) ions at crystal packing interfaces provides a tool to facilitate crystallization of other derivatives. The first crystallographic characterization of As(III) in a biologically relevant tristhiolate coordination environment in the interior of a three-stranded coiled coil reveals the surprising pyramidal coordination of As(III) below the plane of the beta-methylene protons of the three cysteine residues, not above, as was predicted. The parallel orientation of the coiled coil in the presence of metal and the necessity of Zn(II) for crystal packing were confirmed in this 1.8 A structure. Analysis of preferences of Cd(II) and Pb(II) for a vs. d position cysteines in the heptad repeat were evaluated with both monosubstituted and disubstituted peptides. Peptides with one a and one d or two d binding sites were both shown to be able to discriminate between Cd(II) and Pb(II). Fascinatingly, the coordination of Pb(II) was seen to perturb the coordination of Cd(II) to a less favored three-coordinate species from mixed 3- and 4-coordinate species. The investigation of Pb(II) and Cd(II) binding to a peptide containing two a binding sites that was previously shown to have no Cd(II) discrimination resulted in the discovery of a preference for one mixed Pb(II)-Cd(II) species. Results of this work give a structural explanation of spectroscopic and thermodynamic observations, the evaluation of specificity of metal binding for Cd(II) and Pb(II) systems adds to our understanding of the coordination preferences of these toxic metals in biological systems.Ph.D.BiochemistryInorganic chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126840/2/3276314.pd

Data collection and refinement statistics for OprG.

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

Proposed transport mechanism for OmpW family members.

<p>(a) Cartoon of <i>Pseudomonas aeruginosa</i> FadL (PDB ID: 3DWO) viewed from the side <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015016#pone.0015016-Hearn1" target="_blank">[8]</a>. The hatch domain, closing off the barrel on the periplasmic side, is colored red. Bound detergent molecules delineating the hydrophobic transport channel are shown as space-filling models in blue. An arrow marks the lateral opening into the membrane. (b) Surface slab through the center of OprG, showing the hydrophobic channel as a dark tube. Residues Trp170 and Val65, forming the bottom of the channel, are shown in red. An arrow marks the lateral opening into the membrane. (c) Schematic model for transport of small hydrophobic substrates (depicted as octane in green) by members of the OmpW family. The bottom of the channel is shown in red.</p

ClustalW alignment of OprG and other OmpW family members.

<p>The observed secondary structure of OprG is shown above the alignment, with β-strands (S) in blue and the α-helix in loop l3 in red. OprG residues are colored as follows: red; hydrophobic with sidechains pointing inwards, purple; polar/charged with sidechains pointing inwards and green; absolutely conserved prolines lining the lateral opening. The following orthologs have been aligned: Pa, <i>Pseudomonas aeruginosa</i> OprG; Pp, <i>Pseudomonas putida</i> OprG; Ec, <i>E. coli</i> OmpW; Ah, <i>Aeromonas hydrophila</i> OmpW; Vc, <i>Vibrio cholerae</i> OmpW; AlkL, <i>Pseudomonas oleovorans</i> AlkL; DoxH, <i>Pseudomonas</i> sp. (strain C18) DoxH.</p

Structural overview of OprG.

<p>Views from the side (a) and from the extracellular side (b; top panel) and periplasmic side (b; bottom panel). β-strands are colored blue, α-helices red and loops green. Selected extracellular loops are indicated. The approximate positions of the outer membrane interface regions are indicated by horizontal lines. (c) Structural comparison between OprG (blue) and <i>E. coli</i> OmpW (red). Loops have been smoothed for clarity. This and the following figures were made with PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015016#pone.0015016-DeLano1" target="_blank">[32]</a>.</p

In vitro transport assays show a division of Occ channels into two subfamilies.

<p>Uptake of radiolabeled substrates in total membrane vesicles of <i>E. coli</i> Bl21 omp8 expressing empty plasmid (pB22), Occ channels, and <i>E. coli</i> OmpG or FadL. In addition, uptake mediated by the porin-containing strain C43 (DE3) is shown. Substrates are (A) arginine (0.25 µM, 15 min uptake), (B) benzoate (0.5 µM, 10 min), (C) glucuronate (0.5 µM, 10 min), (D) glucose (0.5 µM, 15 min), and (E) pyroglutamate (0.5 µM, 15 min). 100% specific activities correspond to 199.7±5.5 (A), 40.4±0.7 (B), 49.7±1.1 (C), 36.9±1.4 (D), and 20.7±0.5 (E) pmoles substrate/min/mg protein. Filter backgrounds are subtracted from all measurements.</p

Schematic model of transport mediated by OccD1.

<p>Substrate selection occurs by matching of opposite charges (blue, positive; red, negative) present on the substrate and in the constriction of the channel.</p