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Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 Å resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency bothin vitroandin vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G : C→T : A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.

International journal of molecular sciences 22(15), 8315 (1-12) (2021). doi:10.3390/ijms22158315, The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides., Published by Molecular Diversity Preservation International, Basel

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.

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.

Botulinum neurotoxins (BoNTs) are the most potent toxins known. So far, eight serotypes have been identified that all act as zinc-dependent endopeptidases targeting SNARE proteins and inhibiting the release of neurotransmitters. Recently, the first botulinum toxin-like protein was identified outside the Clostridial genus, designated BoNT/Wo in the genome of Weissella oryzae. Here, we report the 1.6 A X-ray crystal structure of the light chain of BoNT/Wo (LC/Wo). LC/Wo presents the core fold common to BoNTs but has an unusually wide, open and negatively charged catalytic pocket, with an additional Ca2+ ion besides the zinc ion and a unique s-hairpin motif. The structural information will help establish the substrate profile of BoNT/Wo and help our understanding of how BoNT evolved.

Due to oncogene expression and altered metabolism, reactive oxygen species (ROS) production is augmented in cancer cells resulting in oxidative DNA damage. 8‑oxoguanine (8-oxoG) is one of the most abundant oxidative DNA lesions. This premutagenic lesion is eliminated from duplex DNA by 8‑Oxoguanine DNA Glycosylase (OGG1), a key player in the base excision repair (BER) pathway. Here, we validate OGG1 as a potential anti-cancer target. OGG1 depletion impairs the growth of A3 T-cell lymphoblastic acute leukemia both in vitro and in vivo, but is well tolerated in non-transformed immortalized cells1. To further validate our findings, we developed TH5487, a potent small-molecule inhibitor that targets OGG1's active site [1,2]. We show that TH5487 suppresses the growth of a wide range of tumor cells, with a favorable therapeutic index compared to non‑transformed cells [1]. Mechanistically, TH5487 treatment inhibits the repair of potassium bromate-induced 8-oxo(d)G lesions, affects OGG1-chromatin dynamics, and hinders OGG1 recruitment to DNA damage regions [3]. Importantly, TH5487 induces replication stress and proliferation arrest1. This study presents a novel mechanistic strategy to exploit ROS elevation in cancer by inhibiting OGG1. References: Visnes, T.; Benitez-Buelga, C.; Cazares-Korner, A.; Sanjiv, K.; Hanna, B.M.; Mortusewicz, O.; Rajagopal, V.; Albers, J.J.; Hagey, D.W.; Bekkhus, T. et al. Targeting OGG1 arrests cancer cell proliferation by inducing replication stress. Nucleic acids research 2020; 48, 12234–12251. Visnes, T.; Cazares-Korner, A.; Hao, W.; Wallner, O.; Masuyer, G.; Loseva, O.; Mortusewicz, O.; Wiita, E.; Sarno, A.; Manoilov, A. et al. Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation. Science 2018, 362, 834–839. Hanna, B.M.F.; Helleday, T.; Mortusewicz, O. OGG1 inhibitor TH5487 alters OGG1 chromatin dynamics and prevents incisions. Biomolecules 2020, 10, 1–10. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by approved by the regional experimental animal Ethical Committee in Stockholm 2010/63 (N8914)

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A–G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a “gain-of-function” mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL–lipid interactions and create a modified BoNT/B with improved therapeutic efficacy., Botulinum neurotoxins are a family of bacterial toxins, some of which are approved for medical and cosmetic uses. This study shows that introducing aromatic residues to a lipid binding loop improved therapeutic efficacy of botulinum neurotoxin B by enhancing its ability to bind to lipid membranes at motor nerve terminals.

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLNE475Q in complex with the products of neurotensin cleavage at 2.7A revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

Tetanus neurotoxin (TeNT) secreted by Clostridium tetani is the causative agent of the spastic paralysis distinctive of human tetanus. TeNT is structurally related to the family of botulinum neurotoxins (BoNTs) produced by Clostridium botulinum that cause flaccid paralysis by disabling synaptic exocytosis at peripheral cholinergic neurons. By contrast, TeNT targets the central nervous system (CNS) by hijacking receptors for neurotrophic factors to enter peripheral neurons thereby being sorted into non‐acidifying endosomes, trafficking via retrograde axonal transport organelles, and entering spinal inhibitory interneurons after transcytosis (Fig 1A). In this issue of EMBO Reports, Masuyer et al 1 describe the structural plasticity of individual TeNT domains in the context of the holotoxin in response to environmental pH, a key factor modulating TeNT fate and action. Through the concerted use of X‐ray crystallography, single particle cryo‐EM, and small angle X‐ray scattering (SAXS), the authors provide snapshots of conformational transitions that may underlie the productive path of TeNT from its entry in the peripheral nervous system (PNS) to its ultimate site of action on central glycinergic synapses.

Clostridial neurotoxins, including tetanus and botulinum neurotoxins, generally target vertebrates. We show here that this family of toxins has a much broader host spectrum, by identifying PMP1, a clostridial-like neurotoxin that selectively targets anopheline mosquitoes. Isolation of PMP1 from Paraclostridium bifermentans strains collected in anopheline endemic areas on two continents indicates it is widely distributed. The toxin likely evolved from an ancestral form that targets the nervous system of similar organisms, using a common mechanism that disrupts SNARE-mediated exocytosis. It cleaves the mosquito syntaxin and employs a unique receptor recognition strategy. Our research has an important impact on the study of the evolution of clostridial neurotoxins and provides the basis for the use of P. bifermentans strains and PMP1 as innovative, environmentally friendly approaches to reduce malaria through anopheline control., So far identified clostridial neurotoxins target vertebrates. Here, Contreras et al. isolate the clostridial-like neurotoxin PMP1 from Paraclostridium bifermentans strains and show that it selectively targets anopheline mosquitoes by targeting mosquito syntaxin.

Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients. Cancer Res; 76(18); 5501–11. ©2016 AACR.

Botulinum neurotoxins (BoNTs) are a family of highly dangerous bacterial toxins, with seven major serotypes (BoNT/A-G). Members of BoNTs, BoNT/A1 and BoNT/B1, have been utilized to treat an increasing number of medical conditions. The clinical trials are ongoing for BoNT/A2, another subtype of BoNT/A, which showed promising therapeutic properties. Both BoNT/A1 and BoNT/A2 utilize three isoforms of synaptic vesicle protein SV2 (SV2A, B, and C) as their protein receptors. We here present a high resolution (2.0 Å) co-crystal structure of the BoNT/A2 receptor-binding domain in complex with the human SV2C luminal domain. The structure is similar to previously reported BoNT/A-SV2C complexes, but a shift of the receptor-binding segment in BoNT/A2 rotates SV2C in two dimensions giving insight into the dynamic behavior of the interaction. Small differences in key residues at the binding interface may influence the binding to different SV2 isoforms, which may contribute to the differences between BoNT/A1 and BoNT/A2 observed in the clinic.

DNA binding as an anti-inflammatory Mice that lack the gene encoding 8-oxoguanine DNA glycosylase 1 (OGG1) show resistance to inflammation. This enzyme binds to sites of oxidative DNA damage and initiates DNA base excision repair. Visnes et al. developed a small-molecule drug that acts as a potent and selective active-site inhibitor that stops OGG1 from recognizing its DNA substrate (see the Perspective by Samson). The drug inhibited DNA repair and modified OGG1 chromatin dynamics, which resulted in the inhibition of proinflammatory pathway genes. The drug was well tolerated by mice and suppressed lipopolysaccharide- and tumor necrosis factor–α–mediated neutrophilic inflammation in the lungs. Science , this issue p. 834 ; see also p. 748

Botulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells., There are seven well-established types of Botulinum neurotoxins (BoNTs). Here the authors report the identification and characterization of a new type of BoNT—BoNT/X—which cleaves a different site on canonical BoNTs substrates and targets SNARE family members not cleaved by known BoNTs.

The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD la can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.

Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. Each BoNT consists of three domains that are essential for toxicity: the binding domain, the translocation domain, and the catalytic light-chain domain. BoNT modular architecture is associated with a multistep mechanism that culminates in the intracellular proteolysis of SNARE (soluble N-ethylmaleimide-sensitive-fusion-protein attachment protein receptor) proteins, which prevents synaptic vesicle exocytosis. As the most toxic proteins known, BoNTs have been extensively studied and are used as pharmaceutical agents to treat an increasing variety of disorders. This review summarizes the level of sophistication reached in BoNT engineering and highlights the diversity of approaches taken to utilize the modularity of the toxin. Improved efficiency and applicability have been achieved by direct mutagenesis and interserotype domain rearrangement. The scope of BoNT activity has been extended to nonneuronal cells and offers the basis for novel biomolecules in the treatment of secretion disorders.

Several soil-derived Actinobacteria produce secondary metabolites that are proven specific and potent inhibitors of the human angiotensin-I-converting enzyme (ACE), a key target for the modulation of hypertension through its role in the renin-angiotensin-aldosterone system. K-26-DCP is a zinc dipeptidyl carboxypeptidase (DCP) produced by Astrosporangium hypotensionis, and an ancestral homologue of ACE. Here we report the high-resolution crystal structures of K-26-DCP and of its complex with the natural microbial tripeptide product K-26. The experimental results provide the structural basis for better understanding the specificity of K-26 for human ACE over bacterial DCPs. Database Structural data are available in the PDB under the accession numbers 5L43 and 5L44.

Human somatic angiotensin-1 converting enzyme (ACE) is a zinc-dependent exopeptidase, that catalyses the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a C-terminal dipeptide. It is the principal component of the renin-angiotensin-aldosterone system that regulates blood pressure. Hence it is an important therapeutic target for the treatment of hypertension and cardiovascular disorders. Here, we report the structures of an ACE homologue from Drosophila melanogaster (AnCE; a proven structural model for the more complex human ACE) co-crystallized with mammalian peptide substrates (bradykinin, Thr(6) -bradykinin, angiotensin I and a snake venom peptide inhibitor, bradykinin-potentiating peptide-b). The structures determined at 2-A resolution illustrate that both angiotensin II (the cleaved product of angiotensin I by AnCE) and bradykinin-potentiating peptide-b bind in an analogous fashion at the active site of AnCE, but also exhibit significant differences. In addition, the binding of Arg-Pro-Pro, the cleavage product of bradykinin and Thr(6) - bradykinin, provides additional detail of the general peptide binding in AnCE. Thus the new structures of AnCE complexes presented here improves our understanding of the binding of peptides and the mechanism by which peptides inhibit this family of enzymes. DATABASE: The atomic coordinates and structure factors for AnCE-Ang II (code 4AA1), AnCE-BPPb (code 4AA2), AnCE-BK (code 4ASQ) and AnCE-Thr6-BK (code 4ASR) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) STRUCTURED DIGITAL ABSTRACT: • AnCE cleaves Ang I by enzymatic study (View interaction) • Bradykinin and AnCE bind by x-ray crystallography (View interaction) • BPP and AnCE bind by x-ray crystallography (View interaction) • AnCE cleaves Bradykinin by enzymatic study (View interaction) • Ang II and AnCE bind by x-ray crystallography (View interaction).

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.

Targeted secretion inhibitors (TSIs) are a new class of engineered biopharmaceutical molecules derived from the botulinum neurotoxins (BoNTs). They consist of the metalloprotease light chain (LC) and translocation domain (Hn) of BoNT; they thus lack the native toxicity towards motor neurons but are able to target soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) proteins. These functional fragment (LHn) derivatives are expressed as single-chain proteins and require post-translational activation into di-chain molecules for function. A range of BoNT derivatives have been produced to demonstrate the successful use of engineered SNARE substrate peptides at the LC–Hn interface that gives these molecules self-activating capabilities. Alternatively, recognition sites for specific exoproteases can be engineered to allow controlled activation. Here, the crystal structures of three LHn derivatives are reported between 2.7 and 3.0 Å resolution. Two of these molecules are derivatives of serotype A that contain a SNARE peptide. Additionally, a third structure corresponds to LHn serotype B that includes peptide linkers at the exoprotease activation site. In all three cases the added engineered segments could not be modelled owing to disorder. However, these structures highlight the strong interactions holding the LHn fold together despite the inclusion of significant polypeptide sequences at the LC–Hn interface.

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACH In binding experiments with 3H-rauwolscine, we studied the interactions of green mamba venom fractions with α2-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α2-adrenoceptors expressed in mammalian cells. KEY RESULTS ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of 3H-rauwolscine binding to the three α2-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α2A-adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA2 values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively. CONCLUSIONS AND IMPLICATIONS ρ-Da1b is the first toxin identified to specifically interact with α2-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α2-adrenoceptor subtypes.

Botulinum neurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocytosis in neurons, at sites distinct from known BoNT cleavage sites on these two proteins. Comparative genomic analysis determines that the E. faecium strain carrying BoNT/En is a commensal type and that the BoNT/En gene is located within a typical BoNT gene cluster on a 206 kb putatively conjugative plasmid. Although the host species targeted by BoNT/En remains to be determined, these findings establish an extended member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multi-drug-resistant (MDR) infections, to horizontally acquire, and possibly disseminate, a unique BoNT gene cluster.

Angiotensin‐1‐converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N‐domain in complex with Aβ fragments. For the physiological Aβ(1–16) peptide, a novel ACE cleavage site was found at His14‐Gln15. Furthermore, Aβ(1–16) was preferentially cleaved by the individual N‐domain; however, the presence of an inactive C‐domain in full‐length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4–10)Q and Aβ(4–10)Y, underwent endoproteolytic cleavage at the Asp7‐Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4–10)Y. Surprisingly, in contrast to Aβ(1–16) and Aβ(4–10)Q, sACE showed positive domain cooperativity and the double C‐domain (CC‐sACE) construct no cooperativity towards Aβ(4–10)Y. The structures of the Aβ peptide–ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C‐terminal P2′ position to the S2′ pocket and recognizes the main chain of the P1′ peptide. It is likely that N‐domain selectivity for the amyloid peptide is conferred through the N‐domain specific S2′ residue Thr358. Additionally, the N‐domain can accommodate larger substrates through movement of the N‐terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full‐length forms. Database The atomic coordinates and structure factors for N‐domain ACE with Aβ peptides 4–10 (5AM8), 10–16 (5AM9), 1–16 (5AMA), 35–42 (5AMB) and (4–10)Y (5AMC) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/).

Angiotensin-I converting enzyme (ACE) is a zinc dipeptidylcarboxypeptidase with two active domains and plays a key role in the regulation of blood pressure and electrolyte homeostasis, making it the principal target in the treatment of cardiovascular disease. More recently, the tetrapetide N-acetyl-Ser–Asp–Lys–Pro (Ac-SDKP) has emerged as a potent antifibrotic agent and negative regulator of haematopoietic stem cell differentiation which is processed exclusively by ACE. Here we provide a detailed biochemical and structural basis for the domain preference of Ac-SDKP. The high resolution crystal structures of N-domain ACE in complex with the dipeptide products of Ac-SDKP cleavage were obtained and offered a template to model the mechanism of substrate recognition of the enzyme. A comprehensive kinetic study of Ac-SDKP and domain co-operation was performed and indicated domain interactions affecting processing of the tetrapeptide substrate. Our results further illustrate the molecular basis for N-domain selectivity and should help design novel ACE inhibitors and Ac-SDKP analogues that could be used in the treatment of fibrosis disorders.

The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3?Å and that of SXN101959 at 3.1?Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B and D. Taken together, these results demonstrate the robustness of the ‘LHn fold’ across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin and serotype D in particular, as a basis for engineering novel secretion inhibitors.

Galectins are evolutionarily conserved and ubiquitously present animal lectins with a high affinity for β-galactose-containing oligosaccharides. To date, 15 mammalian galectins have been identified. Their involvement in cell-cell and cell-matrix interactions has highlighted their importance in signal transduction and other intracellular processes. Human galectin-7 (hGal-7) is a 15 kDa proto type galectin that forms a dimer in solution and its involvement in the stimulation and development of tumour growth has been reported. Previously, we reported the crystal structure of hGal-7 and its complex with galactose and lactose which provided insight into its molecular recognition and detailed interactions. Here, we present newly obtained high-resolution structural data on carbohydrate-based dendrons in complex with hGal-7. Our crystallographic data reveal how multivalent ligands interact with and form cross-links with these galectin molecules. Understanding how these dendrimeric compounds interact with hGal-7 would help in the design of new tools to investigate the recognition of carbohydrates by lectins.

Background: The angiotensin-1-converting enzyme (ACE) is a zinc protease unique in its chloride ion dependence. Results: Key substrate interactions with the catalytic site affect the structure and thermodynamics of ACE, which modulate chloride ion-dependent hydrolysis. Conclusion: Substrate composition influences chloride affinity in ACE. Significance: The interdependence of ACE substrate composition, structure, and thermodynamics influence its chloride ion activation in biology., Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

The K-26 family of bacterial secondary metabolites are N-modified tripeptides terminated by an unusual phosphonate analog of tyrosine. These natural products, produced via three different actinomycetales, are potent inhibitors of human angiotensin-I converting enzyme (ACE). Herein we investigate the interkingdom pharmacology of the K-26 family by synthesizing these metabolites and assessing their potency as inhibitors of both the N-terminal and C-terminal domains of human ACE. In most cases, selectivity for the C-terminal domain of ACE is displayed. Co-crystallization of K-26 in both domains of human ACE reveals the structural basis of the potent inhibition and has shown an unusual binding motif that may guide future design of domain-selective inhibitors. Finally, the activity of K-26 is assayed against a cohort of microbially produced ACE relatives. In contrast to the synthetic ACE inhibitor captopril, which demonstrates broad interkingdom inhibition of ACE-like enzymes, K-26 selectively targets the eukaryotic family.

ACE (angiotensin-1-converting enzyme) is a zinc metallopeptidase that plays a prominent role in blood pressure regulation and electrolyte homeostasis. ACE consists of two homologous domains that despite similarities of sequence and topology display differences in substrate processing and inhibitor binding. The design of inhibitors that selectively inhibit the N-domain (N-selective) could be useful in treating conditions of tissue injury and fibrosis due to build-up of N-domain-specific substrate Ac-SDKP (N-acetyl-Ser–Asp–Lys–Pro). Using a receptor-based SHOP (scaffold hopping) approach with N-selective inhibitor RXP407, a shortlist of scaffolds that consisted of modified RXP407 backbones with novel chemotypes was generated. These scaffolds were selected on the basis of enhanced predicted interaction energies with N-domain residues that differed from their C-domain counterparts. One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay. A molecule incorporating a tetrazole moiety in the P2 position (compound 33RE) displayed potent inhibition (Ki=11.21±0.74 nM) and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of compound 33RE in complex with the N-domain revealed its mode of binding through aromatic stacking with His388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr369. This work further elucidates the molecular basis for N-domainselective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders., The present paper reports the development of an N-domain selective ACE inhibitor and the molecular basis for the interaction of key active site residues with the ligand.

Angiotensin-I converting enzyme (ACE), a two-domain dipeptidylcarboxypeptidase, is a key regulator of blood pressure as a result of its critical role in the renin-angiotensin-aldosterone and kallikrein-kinin systems. Hence it is an important drug target in the treatment of cardiovascular diseases. ACE is primarily known for its ability to cleave angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II), but is also able to cleave a number of other substrates including the vasodilator bradykinin and N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a physiological modulator of hematopoiesis. For the first time we provide a detailed biochemical and structural basis for the domain selectivity of the natural peptide inhibitors of ACE, bradykinin potentiating peptide b and Ang II. Moreover, Ang II showed selective competitive inhibition of the carboxy-terminal domain of human somatic ACE providing evidence for a regulatory role in the human renin-angiotensin system (RAS).