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.

Arsenic, a contaminant of water supplies worldwide, is one of the most toxic inorganic ions. Despite arsenic's health impact, there is relatively little structural detail known about its interactions with proteins. Bacteria such as Escherichia coli have evolved arsenic resistance using the Ars operon that is regulated by ArsR, a repressor protein that dissociates from DNA when As(III) binds. This protein undergoes a critical conformational change upon binding As(III) with three cysteine residues. Unfortunately, structures of ArsR with or without As(III) have not been reported. Alternatively, de novo designed peptides can bind As(III) in an endo configuration within a thiolate-rich environment consistent with that proposed for both ArsR and ArsD. We report the structure of the As(III) complex of Coil Ser L9C to a 1.8-Å resolution, providing x-ray characterization of As(III) in a Tris thiolate protein environment and allowing a structural basis by which to understand arsenated ArsR.arsenic-binding proteins ͉ coiled coil peptides ͉ crystallography ͉ heavy metal toxicity ͉ protein design A rsenic toxicity is a worldwide problem as a natural contaminant of water supplies. Despite the fact that it is a human toxin and carcinogen, few structural reports on its interaction with biological ligands have appeared. Escherichia coli and other bacteria have evolved a detoxification mechanism that employs the arsRDABC operon (1). Arsenic removal by these encoded proteins is initiated when As(III) binds to ArsR, resulting in the dissociation of the repressor protein from the promoter DNA. The structure of the ArsA component of the ATP-dependent extrusion pump ArsAB with antimonite bound in close proximity to the nucleotide-binding site was able to provide some insight into the active transport of As(III) out of the cell (2). Additionally, structural characterization of substrate and product complexes of the arsenate reductase ArsC, which reduces arsenate (AsO 4 3Ϫ ) to arsenite (AsO 2 Ϫ ), has helped elucidate this step of the arsenic detoxification pathway (3, 4). Recent studies of E. coli ArsD indicate that it is a metallochaperone, transporting As(III) to ArsA for extrusion (5). Extended x-ray absorption fine structure and mutagenesis studies have shown that ArsR coordinates As(III) with three cysteine thiolates at a distance of 2.25 Å, a coordination mode that ArsD is also proposed to employ (1, 5). However, no x-ray or NMR structures of ArsR, the repressor protein, or ArsD have been reported to date. Furthermore, AsS 3 structures have not been reported for any biologically relevant small-molecule thiolates, such as glutathione, and only a handful of related AsS 3 complexes with aromatic ligands or chelated alkyl dithiolate coordination are reported (6).We set out to model the putative As(III) coordination environments of ArsR and ArsD in a designed peptide system. Previously, we have shown that the three-stranded coiled coildesigned with the heptad repeat strategy, such as TRI L16C (sequences of all peptides are in Table 1...

Over the past two decades, designed metallopeptides have held the promise for understanding a variety of fundamental questions in metallobiochemistry; however, these dreams have not yet been realized because of a lack of structural data to elaborate the protein scaffolds before metal complexation and the resultant metallated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metallated or nonmetallated forms and no examples where an apo structure and the corresponding metallated peptide assembly have both been defined by x-ray crystallography. Herein we present x-ray structures of two de novo designed parallel three-stranded coiled coils (designed using the heptad repeat (a→g)) CSL9C (CS = Coil Ser) and CSL19C in their non-metallated forms, determined to 1.36 Å and 2.15 Å resolutions, respectively. Leucines from either position 9 (a site) or 19 (d site) are replaced by cysteine to generate the constructs CSL9C and CSL19C, respectively, yielding thiol-rich pockets at the hydrophobic interior of these peptides, suitable to bind heavy metals such as As(III), Hg(II), Cd(II) and Pb(II). We use these structures to understand the inherent structural differences between a and d sites to clarify the basis of the observed differential spectroscopic behavior of metal binding in these types of peptides. Cys side chains of (CSL9C) 3 show alternate † Current Address: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St, Worcester, Massachusetts 01605, USA ‡ Current Address: University of Birmingham, School of Chemistry, Edgbaston, Birmingham, UK B15 2TT Suporting Information Available: Helical wheel diagrams of (CSL9C) 3 , (CSL19C) 3 , location of Zn(II) ions at crystal packing interfaces of (CSL19C) 3 , orientation of Cys Sγ plane with respect to Leu 16 and Leu 23 layers of (CSL19C) 3 , the overlay of α-helical backbones of (CSL9C) 3 , (CSL19C) 3 , (CSL16LPen) 3 and (CSL16DPen) 3 , UV-Vis titrations of Zn(II) to CSL9C and Hg(II), UV-Vis titrations of Hg(II) to CSL9C in the presence and absence of Zn(II) and 199 Hg NMR spectra of CSL9C with 199 Hg(II) in the presence and absence of Zn(II). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 September 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C) 3 are present as a single conformer. Zn(II) ions, which do not coordinate or influence Cys residues at the designed metal sites but are essential for forming x-ray quality crystals, are bound to His and Glu residues at the crystal packing interfaces of both structures. These "apo" structures are used to clarify the changes in metal site organization between metallated As(CSL9C) 3 and to speculate on the differential basis of Hg(II) binding in a versus d peptid...

BackgroundThe 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 FindingsIn 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 Å resolution. OprG forms an eight-stranded β-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/SignificanceThe 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 lateral diffusion mechanism similar to that of E. coli FadL.

Exudative age-related macular degeneration (AMD) is the most common cause of moderate and severe vision loss in developed countries.