Structure of dual receptor binding to botulinum neurotoxin B

Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3 Å structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor Synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards Synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points

Botulinum and tetanus neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering

Crystal Structure of Human Cytosolic 5′-Nucleotidase II INSIGHTS INTO ALLOSTERIC REGULATION AND SUBSTRATE RECOGNITION

Cytosolic 5′-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adenine-based nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5′-nucleotidase II as native protein (2.2 A) and in complex with adenosine (1.5A) and beryllium trifluoride (2.15A). The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5′(3′)-deoxyribonucleotidase and cytosolic 5′-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5′-nucleotidase II with that of mitochondrial 5′(3′)-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition

Structural basis for the interaction of the chaperone Cbp3 with newly synthesized cytochrome b during mitochondrial respiratory chain assembly

Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3-Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol-cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein

Structural studies of tri-functional human GART

Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway

Crystal structure of the P2 C-repressor: a binder of non-palindromic direct DNA repeats

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor

Increased Levels of Inflammatory Cytokines and Endothelin-1 in Alveolar Macrophages from Patients with Chronic Heart Failure

BACKGROUND: Pathophysiological interactions between heart and lungs in heart failure (HF) are well recognized. We investigated whether expression of different factors known to be increased in the myocardium and/or the circulation in HF is also increased in alveolar macrophages in HF. METHODOLOGY/PRINCIPAL FINDINGS: Lung function, hemodynamic parameters, gene expression in alveolar macrophages, and plasma levels in the pulmonary and femoral arteries of HF patients (n = 20) were compared to control subjects (n = 16). Our principal findings were: (1) Lung function was significantly lower in HF patients compared to controls (P<0.05). (2) mRNA levels of ET-1, tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) were increased in alveolar macrophages from HF patients. (3) Plasma levels of ET-1, TNFα, IL-6 and MCP-1 were significantly increased in HF patients, whereas our data indicate a net pulmonary release of MCP-1 into the circulation in HF. CONCLUSIONS/SIGNIFICANCE: Several important cytokines and ET-1 are induced in alveolar macrophages in human HF. Further studies should clarify whether increased synthesis of these factors affects pulmonary remodeling and, directly or indirectly, adversely affects the failing myocardium

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment