J. van der Oost

Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors.

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, γ-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR α-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined ‘loops’ A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer’s disease and nicotine addiction.

The CRISPRs, They Are A-Changin': How Prokaryotes Generate Adaptive Immunity

All organisms need to continuously adapt to changes in their environment. Through horizontal gene transfer, bacteria and archaea can rapidly acquire new traits that may contribute to their survival. However, because new DNA may also cause damage, removal of imported DNA and protection against selfish invading DNA elements are also important. Hence, there should be a delicate balance between DNA uptake and DNA degradation. Here, we describe prokaryotic antiviral defense systems, such as receptor masking or mutagenesis, blocking of phage DNA injection, restriction/modification, and abortive infection. The main focus of this review is on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated), a prokaryotic adaptive immune system. Since its recent discovery, our biochemical understanding of this defense system has made a major leap forward. Three highly diverse CRISPR/Cas types exist that display major structural and functional differences in their mode of generating resistance against invading nucleic acids. Because several excellent recent reviews cover all CRISPR subtypes, we mainly focus on a detailed description of the type I-E CRISPR/Cas system of the model bacterium Escherichia coli K12.

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positiveDesulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a V max of 28 units/mg protein and a K m of 20 μm. The pH and temperature optimum were 8.2 and 52 °C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E m ) = −440 mV), one [3Fe-4S] cluster (E m = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an E m of −370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

Alternative affinity tools: more attractive than antibodies?

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.

Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus.

We report here the first molecular characterization of an endo-β-1,3-glucanase from an archaeon.Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-β-1,3- and endo-β-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, alamA fragment encoding an active endo-β-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the β-1,3-glucose polymer laminarin and has some hydrolytic activity on the β-1,3-1,4 glucose polymers lichenan and barley β-glucan. The enzyme is the most thermostable endo-β-1,3-glucanase described up to now; it has optimal activity at 100–105 °C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-β-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitrohydrolysis of laminarin by the endo-β-1,3-glucanase proceeds more efficiently in combination with an exo-β-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on β-glucans.

FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation

The Paracoccus denitrificansfnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high‐affinity cbb3‐type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox‐sensitive [4Fe–4S] cluster in FNR. NNR, another FNR‐like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane‐bound nitrate reductase and the cbb3‐type oxidase was significantly reduced, whilst the activity of the bb3‐type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3‐type oxidase, cytochrome c550, cytochrome c552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP‐ and NNR‐regulated genes are highly similar to or even identical to each other, the absence of cross‐talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.

Transcriptional regulation of the cpr gene cluster in ortho-chlorophenol-respiring Desulfitobacterium dehalogenans.

To characterize the expression and possible regulation of reductive dehalogenation in halorespiring bacteria, a 11.5-kb genomic fragment containing the o-chlorophenol reductive dehalogenase-encoding cprBA genes of the gram-positive bacterium Desulfitobacterium dehalogenans was subjected to detailed molecular characterization. Sequence analysis revealed the presence of eight designated genes with the order cprTKZEBACD and with the same polarity except for cprT. The deduced cprC and cprK gene products belong to the NirI/NosR and CRP-FNR families of transcription regulatory proteins, respectively. CprD and CprE are predicted to be molecular chaperones of the GroEL type, whereas cprT may encode a homologue of the trigger factor folding catalysts. Northern blot analysis, reverse transcriptase PCR, and primer extension analysis were used to elucidate the transcriptional organization and regulation of the cpr gene cluster. Results indicated halorespiration-specific transcriptional induction of the monocistronic cprT gene and the biscistronic cprBA and cprZE genes. Occasional read-through at cprC gives rise to a tetracistronic cprBACD transcript. Transcription of cprBA was induced 15-fold upon addition of the o-chlorophenolic substrate 3-chloro-4-hydroxyphenylacetic acid within 30 min with concomitant induction of dehalogenation activity. Putative regulatory protein binding motifs that to some extent resemble the FNR box were identified in the cprT-cprK and cprK-cprZ intergenic regions and the promoter at cprB, suggesting a role for FNR-like CprK in the control of expression of the cprTKZEBACD genes.

Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome ofP. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 °C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K m values for fructose 6-phosphate and ADP were 2.3 and 0.11 mm, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising theK m for ADP to 0.34 and 0.41 mm, respectively.

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage.

Significance We have solved crystal structures of ternary Thermus thermophilus Argonaute (Ago) complexes with guide and target DNA in cleavage-incompatible, cleavage-compatible, and postcleavage states in the 2.2- to 2.3-Å resolution range, thereby identifying the relative positions of catalytic residues, a pair of Mg2+ cations, and the nucleophilic water poised for in-line attack on the cleavable phosphate. These higher resolution structures represent snapshots of distinct key steps in the catalytic RNase H-mediated cleavage pathway, providing additional detailed insights into Ago-mediated cleavage chemistry of target strands. Importantly, a Glu residue shifts from an “outside” to an “inside” conformation where it inserts into the catalytic pocket to complete a catalytic tetrad during the transition from a cleavage-incompatible to a cleavage-compatible conformation. We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome

Guillain–Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.