Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM(4-) siderophore analogues of varied linker length

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM(4-)) to five, six and eight (5-, 6-, 8-LICAM(4-), respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM(4-) structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM(4-) is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM(4-) but decreases for 6- and 8-LICAM(4-). The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288

Structural and Sequence Analysis of Imelysin-Like Proteins Implicated in Bacterial Iron Uptake

Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution), have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function

Accessing a Hidden Conformation of the Maltose Binding Protein Using Accelerated Molecular Dynamics

Periplasmic binding proteins (PBPs) are a large family of molecular transporters that play a key role in nutrient uptake and chemotaxis in Gram-negative bacteria. All PBPs have characteristic two-domain architecture with a central interdomain ligand-binding cleft. Upon binding to their respective ligands, PBPs undergo a large conformational change that effectively closes the binding cleft. This conformational change is traditionally viewed as a ligand induced-fit process; however, the intrinsic dynamics of the protein may also be crucial for ligand recognition. Recent NMR paramagnetic relaxation enhancement (PRE) experiments have shown that the maltose binding protein (MBP) - a prototypical member of the PBP superfamily - exists in a rapidly exchanging (ns to µs regime) mixture comprising an open state (approx 95%), and a minor partially closed state (approx 5%). Here we describe accelerated MD simulations that provide a detailed picture of the transition between the open and partially closed states, and confirm the existence of a dynamical equilibrium between these two states in apo MBP. We find that a flexible part of the protein called the balancing interface motif (residues 175–184) is displaced during the transformation. Continuum electrostatic calculations indicate that the repacking of non-polar residues near the hinge region plays an important role in driving the conformational change. Oscillations between open and partially closed states create variations in the shape and size of the binding site. The study provides a detailed description of the conformational space available to ligand-free MBP, and has implications for understanding ligand recognition and allostery in related proteins

A Novel Extracytoplasmic Function (ECF) Sigma Factor Regulates Virulence in Pseudomonas aeruginosa

Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a β-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen

Mutational Analysis of Hemoglobin Binding and Heme Utilization by a Bacterial Hemoglobin Receptor

Iron is an essential nutrient for most living organisms. To acquire iron from their environment, Gram-negative bacteria use TonB-dependent transporters that bind host proteins at the bacterial surface and transport iron or heme to the periplasm via the Ton machinery. TonB-dependent transporters are barrel-shaped outer membrane proteins with 22 transmembrane domains, 11 surface-exposed loops, and a plug domain that occludes the pore. To identify key residues of TonB-dependent transporters involved in hemoglobin binding and heme transport and thereby locate putative protective epitopes, the hemoglobin receptor of Haemophilus ducreyi HgbA was used as a model of iron/heme acquisition from hemoglobin. Although all extracellular loops of HgbA are required by H. ducreyi to use hemoglobin as a source of iron/heme, we previously demonstrated that hemoglobin binding by HgbA only involves loops 5 and 7. Using deletion, substitution, and site-directed mutagenesis, we were able to differentiate hemoglobin binding and heme acquisition by HgbA. Deletion or substitution of the GYEAYNRQWWA region of loop 5 and alanine replacement of selected histidines affected hemoglobin binding by HgbA. Conversely, mutation of the phenylalanine in the loop 7 FRAP domain or substitution of the NRQWWA motif of loop 5 significantly abrogated utilization of heme from hemoglobin. Our findings show that hemoglobin binding and heme utilization by a bacterial hemoglobin receptor involve specific motifs of HgbA

Fate of trace metals in anaerobic digestion

© Springer International Publishing Switzerland 2015. A challenging, and largely uncharted, area of research in the field of anaerobic digestion science and technology is in understanding the roles of trace metals in enabling biogas production. This is a major knowledge gap and a multifaceted problem involving metal chemistry; physical interactions of metal and solids; microbiology; and technology optimization. Moreover, the fate of trace metals, and the chemical speciation and transport of trace metals in environments— often agricultural lands receiving discharge waters from anaerobic digestion processes— simultaneously represents challenges for environmental protection and opportunities to close process loops in anaerobic digestion.The authors acknowledge funding within the framework of the COST Action 1302 (‘European Network on Ecological Roles of Trace Metals in Anaerobic Biotechnologies’). GC is supported by a European Research Council Starting Grant (‘3C-BIOTECH; No. 261330).Peer Reviewe