Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding.

The ring-shaped cohesin complex is a key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is hindered by Smc3 acetylation.

The Stringent Response Inhibits 70S Ribosome Formation in by Impeding GTPase-Ribosome Interactions.

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states.

The structural basis for high-affinity uptake of lignin-derived aromatic compounds by proteobacterial TRAP transporters.

The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the β-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals.

Characterisation of a tripartite α-pore forming toxin from Serratia marcescens.

Tripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S.

The molecular basis of endolytic activity of a multidomain alginate lyase from , a representative of a new lyase family, PL39.

Alginate is a polymer containing two uronic acid epimers, β-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multimodular, broad-specificity endotype alginate lyase from the marine thermophile , which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PL39.

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The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC).

A New Mechanism for High-Affinity Uptake of C4-Dicarboxylates in Bacteria Revealed by the Structure of Rhodopseudomonas palustris MatC (RPA3494), a Periplasmic Binding Protein of the Tripartite Tricarboxylate Transporter (TTT) Family.

C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake.

Structural basis for high-affinity adipate binding to AdpC (RPA4515), an orphan periplasmic-binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris.

Unlabelled: The tripartite tricarboxylate transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate-binding protein (SBP) for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP-ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with K values in the μm range.

Direct observation of DNA threading in flap endonuclease complexes.

Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.

Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.

Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function.

Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry.

Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft.

The CouPSTU and TarPQM transporters in Rhodopseudomonas palustris: redundant, promiscuous uptake systems for lignin-derived aromatic substrates.

The biodegradation of lignin, one of the most abundant carbon compounds on Earth, has important biotechnological applications in the derivation of useful products from lignocellulosic wastes. The purple photosynthetic bacterium Rhodopseudomonas palustris is able to grow photoheterotrophically under anaerobic conditions on a range of phenylpropeneoid lignin monomers, including coumarate, ferulate, caffeate, and cinnamate. RPA1789 (CouP) is the periplasmic binding-protein component of an ABC system (CouPSTU; RPA1789, RPA1791-1793), which has previously been implicated in the active transport of this class of aromatic substrate.

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase.

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli.

Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, from Burkholderia pseudomallei.

bpsl0128, a gene encoding a putative response regulator from Burkholderia pseudomallei strain D286, has been cloned into a pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. The full-length protein is degraded during purification to leave a fragment corresponding to the putative receiver domain, and crystals of this protein that diffracted to beyond 1.75 Å resolution have been grown by the hanging-drop vapour-diffusion technique using PEG 6000 as the precipitant.

The stalk region of the RecU resolvase is essential for Holliday junction recognition and distortion.

The Bacillus subtilis RecU protein has two activities: to recognize, distort, and cleave four-stranded recombination intermediates and to modulate RecA activities. The RecU structure shows a mushroom-like appearance, with a cap and a stalk region. The RuvB interaction and the catalytic residues are located in the cap region of dimeric RecU.

The virulence factor PEB4 (Cj0596) and the periplasmic protein Cj1289 are two structurally related SurA-like chaperones in the human pathogen Campylobacter jejuni.

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.

Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents.

Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P.

Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates.

A knowledge of the structures of acyl chain loaded species of the acyl carrier protein (ACP) as used in fatty acid biosynthesis and a range of other metabolic events, is essential for a full understanding of the molecular recognition at the heart of these processes. To date the only crystal structure of an acylated species of ACP is that of a butyryl derivative of Escherichia coli ACP. We have now determined the structures of a family of acylated E.

RusA Holliday junction resolvase: DNA complex structure--insights into selectivity and specificity.

We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli RusA resolvase bound to a duplex DNA substrate that reveals critical protein-DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The RusA enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, RusA shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA.

Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family.

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family.

The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold.

Holliday junction resolution performed by a variety of structure-specific endonucleases is a key step in DNA recombination and repair. It is believed that all resolvases carry out their reaction chemistries in a similar fashion, utilizing a divalent cation to facilitate the hydrolysis of the phosphodiester backbone of the DNA, but their architecture varies. To date, with the exception of bacteriophage T4 endonuclease VII, each of the known resolvase enzyme structures has been categorized into one of two families: the integrases and the nucleases.

Crystallization of RusA Holliday junction resolvase from Escherichia coli.

Crystals of the Escherichia coli Holliday junction resolvase RusA have been obtained using the hanging-drop method and characterized. The crystals have a primitive monoclinic form and belong to space group P2(1). The V(M) value suggests the presence of two copies of the monomer in the asymmetric unit.

Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography.

The RuvABC resolvasome of Escherichia coli typifies nucleoprotein complexes involved in genetic transactions. This molecular assembly catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease.

Expression, purification and crystallization of the Plasmodium falciparum enoyl reductase.

New hope has been gained in the control of the malaria parasite Plasmodium falciparum (pf) with the discovery that the parasite contains a prokaryotic type II fatty-acid synthase (FAS). Since enzymes of this type are absent in humans, they are potential targets for the development of new drugs. The enoyl reductase enzyme (ENR) belonging to this pathway is of particular interest because it has been shown to be inhibited by submicromolar concentrations of the antimicrobial agent triclosan.

Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens.

Synercid, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.