Wulf Blankenfeldt

The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rab1b.

Legionella Hijacks Rab Legionella pneumophila can infect eukaryotic cells and takes up residence within intracellular vacuoles, where it multiplies. In order to produce and maintain this intracellular niche, the pathogen must manipulate membrane trafficking within the host cell. Now, Müller et al. (p. 946, published online 22 July) describe the ability of Legionella pneumophila to manipulate vesicular trafficking by the covalent modification of the small guanosine triphosphatase (GTPase) Rab1, which normally regulates the transport of endoplasmic reticulum–derived vesicles in eukaryotic cells. The Legionella protein DrrA is released into the cytosol of infected cells, where it specifically AMPylates a tyrosine residue of one of the regulating regions of Rab1. The modification renders the Rab protein inaccessible to GTPase-activating proteins and thus locks it in its active guanosine triphosphate–bound state. An intracellular bacterial pathogen interferes with host cell membrane trafficking. In the course of Legionnaires’ disease, the bacterium Legionella pneumophila affects the intracellular vesicular trafficking of infected eukaryotic cells by recruiting the small guanosine triphosphatase (GTPase) Rab1 to the cytosolic face of the Legionella-containing vacuole. In order to accomplish this, the Legionella protein DrrA contains a specific guanine nucleotide exchange activity for Rab1 activation that exchanges guanosine triphosphate (GTP) for guanosine diphosphate on Rab1. We found that the amino-terminal domain of DrrA possesses adenosine monophosphorylation (AMPylation) activity toward the switch II region of Rab1b, leading to posttranslational covalent modification of tyrosine 77. AMPylation of switch II by DrrA restricts the access of GTPase activating proteins, thereby rendering Rab1b constitutively active.

The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA)

The synthesis of deoxy‐thymidine di‐phosphate (dTDP)–L‐rhamnose, an important component of the cell wall of many microorganisms, is a target for therapeutic intervention. The first enzyme in the dTDP–L‐rhamnose biosynthetic pathway is glucose‐1‐phosphate thymidylyltransferase (RmlA). RmlA is inhibited by dTDP–L‐rhamnose thereby regulating L‐rhamnose production in bacteria. The structure of Pseudomonas aeruginosa RmlA has been solved to 1.66 Å resolution. RmlA is a homotetramer, with the monomer consisting of three functional subdomains. The sugar binding and dimerization subdomains are unique to RmlA‐like enzymes. The sequence of the core subdomain is found not only in sugar nucleotidyltransferases but also in other nucleotidyltransferases. The structures of five distinct enzyme substrate–product complexes reveal the enzyme mechanism that involves precise positioning of the nucleophile and activation of the electrophile. All the key residues are within the core subdomain, suggesting that the basic mechanism is found in many nucleotidyltransferases. The dTDP–L‐rhamnose complex identifies how the protein is controlled by its natural inhibitor. This work provides a platform for the design of novel drugs against pathogenic bacteria.

RabGDI Displacement by DrrA from Legionella Is a Consequence of Its Guanine Nucleotide Exchange Activity

Prenylated Rab proteins exist in the cytosol as soluble, high-affinity complexes with GDI that need to be disrupted for membrane attachment and targeting of Rab proteins. The Legionella pneumophila protein DrrA displaces GDI from Rab1:GDI complexes, incorporating Rab1 into Legionella-containing vacuoles and activating Rab1 by exchanging GDP for GTP. Here, we present the crystal structure of a complex between the GEF domain of DrrA and Rab1 and a detailed kinetic analysis of this exchange. DrrA efficiently catalyzes nucleotide exchange and mimics the general nucleotide exchange mechanism of mammalian GEFs for Ras-like GTPases. We show that the GEF activity of DrrA is sufficient to displace prenylated Rab1 from the Rab1:GDI complex. Thus, apparent GDI displacement by DrrA is linked directly to nucleotide exchange, suggesting a basic model for GDI displacement and specificity of Rab localization that does not require discrete GDI displacement activity.

Diversity and Evolution of the Phenazine Biosynthesis Pathway

ABSTRACT Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.

Noncompaction of the Ventricular Myocardium Is Associated with a De Novo Mutation in the β-Myosin Heavy Chain Gene

Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the α- and β-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebsteins anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium.

Analysis of the eukaryotic prenylome by isoprenoid affinity tagging

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein

Information processing pathways such as DNA replication are conserved in eukaryotes and archaea and are significantly different from those found in bacteria. Single‐stranded DNA‐binding (SSB) proteins (or replication protein A, RPA, in eukaryotes) play a central role in many of these pathways. However, whilst euryarchaea have a eukaryotic‐type RPA homologue, crenarchaeal SSB proteins appear much more similar to the bacterial proteins, with a single OB fold for DNA binding and a flexible C‐terminal tail that is implicated in protein–protein interactions. We have determined the crystal structure of the SSB protein from the crenarchaeote Sulfolobus solfataricus to 1.26 Å. The structure shows a striking and unexpected similarity to the DNA‐binding domains of human RPA, providing confirmation of the close relationship between archaea and eukaryotes. The high resolution of the structure, together with thermodynamic and mutational studies of DNA binding, allow us to propose a molecular basis for DNA binding and define the features required for eukaryotic and archaeal OB folds.

Of Two Make One: The Biosynthesis of Phenazines

Physicians of the 19th century were familiar with the conspicuous occurrence of “blue pus”, which they sometimes observed in patients with severe purulent wounds. Even older are reports of and folk remedies against “blue milk”, a coloration that sometimes developed in fresh milk after some days. Key insight into these phenomena was provided in 1859—exactly 150 years ago—when Mathurin-Joseph Fordos, at a session of the Societ d’ mulation pour les Sciences Pharmaceutiques, reported the isolation of the blue pigment “pyocyanine” (from the Greek words for “pus” and “blue”; pyocyanine is nowadays more commonly spelled as pyocyanin) from purulent wound dressings by chloroform extraction. Pyocyanin (5-N-methyl-1hydroxophenazinium betaine) was the first example of a phenazine natural product, a compound class that has grown to well over 100 members since this first report. Due to the improved understanding of their importance to the phenazinegenerating and also to commensal species, the phenazines have been reviewed several times in recent years. Here, we provide a historical perspective of the more than 100 years of research that led us to our current picture of the interesting biosynthesis of phenazine natural products. The details of Fordos’ pyocyanin isolation method, chloroform extraction followed by acidification and partition into an aqueous phase, were published one year later and are still in use today, but it took until 1882 for the French pharmacist Carle Gessard to show that the blue coloration in pus was due to the presence of a microorganism that he then termed Bacillus pyocyaneus. B. pyocyaneus is nowadays known as Pseudomonas aeruginosa, and the Latin term still reflects this strain’s capacity to secrete colored compounds in the modern name: “aerugo” is the Latin word for verdigris, the blue–green coating that develops on copper after long exposure to air. P. aeruginosa is an important human opportunistic pathogen responsible for a large number of nosocomial infections, and it is also the main course of low life expectancy in patients with cystic fibrosis due to chronic infections of the lungs. The production of pyocyanin is used both for identification in the clinic and as a reporter signal in P. aeruginosa research until today. The occurrence of blue milk, on the other hand, is probably due to an environmental strain of P. fluorescens, and it is not yet clear if this coloration also is a consequence of phenazine production. Gessard’s discovery of P. aeruginosa was resonated in many publications from the medical field, but it required more than 50 years before the correct chemical structure of pyocyanin was established. The chemical composition was first studied by Ledderhose, who derived a formula that was later corrected by McCombie and Scarborough and by Wrede and Strack. Wrede and Strack were also the first to discover a phenazine moiety in a breakdown product of pyocyanin, but their studies were hampered by the fact that they could only obtain a defined molecular weight when working in glacial acetic acid, under which circumstances they obtained a pyocyanin dimer. This dimer was questioned by the results of electrochemical studies by Elema and by Friedheim and Michaelis, before Hillemann finally derived the correct structure in 1938. In retrospect, it seems possible that the conditions employed by Wrede and Strack induced a 1:1 charge-transfer complex of reduced and oxidized pyocyanin, similar to the phenazine derivative chlororaphin, which is also produced by P. aeruginosa (Figure 1). Jensen and Holten later measured the dipole moment of pyocyanin and found that its zwitterionic mesomer is present in considerable amounts. In the course of these studies, it became clear that pyocyanin is a redox-active compound that changes its color depending on pH and oxidation state. This also explained the “chameleon phenomenon”, which describes a temporary color change of P. aeruginosa cultures on solid media after exposure to air by disturbance with a platinum needle. Since the first isolation by Fordos, more than 100 phenazine derivatives modified at all positions of the ring system and colored in all shades of

Structures of the dual bromodomains of the P-TEFb activating protein Brd4 at atomic resolution

Brd4 is a member of the bromodomains and extra terminal domain (BET) family of proteins that recognize acetylated chromatin structures through their bromodomains and act as transcriptional activators. Brd4 functions as an associated factor and positive regulator of P-TEFb, a Cdk9-cyclin T heterodimer that stimulates transcriptional elongation by RNA polymerase II. Here, the crystal structures of the two bromodomains of Brd4 (BD1 and BD2) were determined at 1.5 and 1.2 Å resolution, respectively. Complex formation of BD1 with a histone H3 tail polypeptide encompassing residues 12–19 showed binding of the Nζ-acetylated lysine 14 to the conserved asparagine 140 of Brd4. In contrast, in BD2 the N-terminal linker sequence was found to interact with the binding site for acetylated lysines of the adjacent molecule to form continuous strings in the crystal lattice. This assembly shows for the first time a different binding ligand than acetylated lysine indicating that also other sequence compositions may be able to form similar interaction networks. Isothermal titration calorimetry revealed best binding of BD1 to H3 and of BD2 to H4 acetylated lysine sequences, suggesting alternating histone recognition specificities. Intriguingly, an acetylated lysine motif from cyclin T1 bound similarly well to BD2. Whereas the structure of Brd2 BD1 suggested its dimer formation, both Brd4 bromodomains appeared monomeric in solution as shown by size exclusion chromatography and mutational analyses.

Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp.

Phenazine compounds represent a large class of bacterial metabolites that are produced by some fluorescent Pseudomonas spp. and a few other bacterial genera. Phenazines were first noted in the scientific literature over 100 years ago, but for a long time were considered to be pigments of uncertain function. Following evidence that phenazines act as virulence factors in the opportunistic human and animal pathogen Pseudomonas aeruginosa and are actively involved in the suppression of plant pathogens, interest in these compounds has broadened to include investigations of their genetics, biosynthesis, activity as electron shuttles, and contribution to the ecology and physiology of the cells that produce them. This minireview highlights some recent and exciting insights into the diversity, frequency and ecological roles of phenazines produced by fluorescent Pseudomonas spp.