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Shigella dysentriae and other Gram-negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 Å resolution the 3D structure of the TonB-dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C-terminal domain that folds into a 22-stranded transmembrane β-barrel, which is filled by the N-terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 Å apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA-Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N-terminal TonB-box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB-box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB-box. Proteins 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 Å resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 Å resolution using one crystal of ShuA-Pb, and at 3.2 Å resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL. [ABSTRACT FROM AUTHOR]

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonBdependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed. [ABSTRACT FROM AUTHOR]

Ferripyoverdine transport across the outer membrane of Pseudomonas aeruginosa by the pyoverdine receptor FpvA and the transcriptional regulation of FpvA involve interactions of the FpvA N-terminal TonB box and signalling domain with proteins from the inner membrane. Several crystallization conditions of FpvA–Pvd-Fe solubilized in C8E4 detergent were obtained and X-ray data were collected from three crystal forms. The resolution limits range from 3.15 to 2.7 Å depending on the crystal form. From preliminary analysis of the electron-density maps, the first full-length structure of an outer membrane receptor including a signalling domain should be determined. [ABSTRACT FROM AUTHOR]

Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc 1 complex. Structure–activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pK a for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc 1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc 1 complex at 2.28Å resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the αA helix. [Copyright &y& Elsevier]

Peptide methionine sulfoxide reductase mediates the reduction of protein sulfoxide methionyl residues back to methionines and could thus be implicated in the antioxidant defence of organisms. Hexagonal crystals of the Escherichia coli enzyme (MsrA) were obtained by the hanging-drop vapour-diffusion technique. They belong to space group P6522, with unit-cell parameters a = b = 102.5, c = 292.3 Å, γ = 120°. A native data set was collected at 1.9 Å resolution. Crystals of selenomethionine-substituted MsrA were also grown under the same crystallization conditions. A three-wavelength MAD experiment has led to the elucidation of the positions of the Se atoms and should result in a full structure determination. [ABSTRACT FROM AUTHOR]

FptA, the pyochelin outer membrane receptor from Pseudomonas aeruginosa, is a siderophore receptor involved in iron uptake when the bacterium grows under iron limitation. Two crystal forms of the FptA-pyochelin complex were obtained under different crystallization conditions. They belong to space groups P1 and P212121 and data sets were collected for both crystal forms. The triclinic crystals diffract to 3.2 Å resolution and the orthorhombic crystals show a 1.9 Å resolution limit. A data set at the peak of the iron K edge was also collected at 3.1 Å resolution. [ABSTRACT FROM AUTHOR]

FpvA, the pyoverdine outer-membrane receptor from Pseudomonas aeruginosa, is involved in iron uptake when bacteria grow under iron limitation. Crystals of the in vivo pyoverdine-loaded FpvA were obtained under several crystallization conditions using different detergents. A native data set was collected at 3.6 Å resolution and a three-wavelength MAD data set was collected at 3.6 Å resolution using crystals of selenomethionine-substituted protein. The crystals grew under similar conditions and both belong to space group C2, but have different unit-cell parameters. [ABSTRACT FROM AUTHOR]

Due to the rising incidence of antibiotic-resistant infections, the last-line antibiotics, polymyxins, have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin B by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd ( arn ) operon. In this work, we characterized a polymyxin-induced operon named mipBA , present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by the ParR/ParS two-component regulatory system in response to polymyxins. Structural modeling revealed that MipA folds as an outer-membrane β-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule, while the lipoprotein MipB adopts a β-lactamase fold with two additional C-terminal domains. Experimental work confirmed that MipA and MipB localize to the bacterial envelope, and they co-purify in vitro . Nano differential scanning fluorimetry showed that polymyxins stabilized MipA in a specific and dose-dependent manner. Mass spectrometry-based quantitative proteomics on P. aeruginosa membranes demonstrated that ∆ mipBA synthesized fourfold less MexXY-OprA proteins in response to polymyxin B compared to the wild-type strain. The decrease was a direct consequence of impaired transcriptional activation of the mex operon operated by ParR/ParS. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pump, MexXY-OprA.IMPORTANCEDue to the emergence of multidrug-resistant isolates, antibiotic options may be limited to polymyxins to eradicate Gram-negative infections. Pseudomonas aeruginosa , a leading opportunistic pathogen, has the ability to develop resistance to these cationic lipopeptides by modifying its lipopolysaccharide through proteins encoded within the arn operon. Herein, we describe a sub-group of P. aeruginosa strains lacking the arn operon yet exhibiting adaptability to polymyxins. Exposition to sub-lethal polymyxin concentrations induced the expression and production of two envelope-associated proteins. Among those, MipA, an outer-membrane barrel, is able to specifically bind polymyxins with an affinity in the 10-µM range. Using membrane proteomics and phenotypic assays, we showed that MipA and MipB participate in the adaptive response to polymyxins via ParR/ParS regulatory signaling. We propose a new model wherein the MipA-MipB module functions as a novel polymyxin sensing mechanism., Competing Interests: The authors declare no conflict of interest.

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.