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Made available in DSpace on 2022-04-28T19:44:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-30 Medical Research Council Australian Research Council Endoparasitoid wasps use complex biochemical arsenals to suppress the normal humoral and cellular immune responses of their hosts in order to transform them into a suitable environment for development of their eggs and larvae. Venom injected during oviposition is a key component of this arsenal, but the functions of individual venom toxins are still poorly understood. Furthermore, there has been little investigation of the potential biotechnological use of these venom toxins, for example for control of agricultural pests. The endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) is a biocontrol agent reared in biofactories and released extensively in Brazil to control the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). The objectives of this work were to reveal venom components produced by C. flavipes and explore the function of a major venom peptide, Cf4. Using a combined proteomic/transcriptomic approach, we identified 38 putative venom toxins including both linear and disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins, and proteins of the odorant binding families. Because of its high abundance in the venom, we selected Cf4, a 33-residue peptide with three disulfide bonds, for synthesis and further characterization. We found that synthetic Cf4 reduced the capacity of D. saccharalis hemocytes to encapsulate foreign bodies without any effect on phenoloxidase activity, consistent with a role in disruption of the cellular host immune response. Feeding leaves coated with Cf4 to neonate D. saccharalis resulted in increased mortality and significantly reduced feeding compared to caterpillars fed untreated leaves, indicating that Cf4 is a potential candidate for insect pest control through ingestion. This study adds to our knowledge of endoparasitoid wasp venoms composition, host regulation mechanisms and their biotechnological potential for pest management. School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Institute for Molecular Bioscience The University of Queensland Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science The University of Queensland School of Agricultural and Veterinarian Sciences São Paulo State University (UNESP) Australian Research Council: CE200100012

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma.We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish).Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFWe unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.

α-Conotoxins that target muscle nicotinic acetylcholine receptors (nAChRs) commonly fall into two structural classes, frameworks I and II containing two and three disulfide bonds, respectively. Conotoxin SII is the sole member of the cysteine-rich framework II with ill-defined interactions at the nAChRs. Following directed synthesis of α-SII, NMR analysis revealed a well-defined structure containing a 3

Significance Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.

Voltage-gated sodium (NaV) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The NaV1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of SCN1A, the gene encoding NaV1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of NaV1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of NaV1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of NaV channels, but little is known about their activity at NaV1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of NaV1.1 from venom of the buthid scorpion Hottentotta jayakari. These peptides, Hj1a and Hj2a, are potent agonists of NaV1.1 (EC50 of 17 and 32 nM, respectively), and they present dual α/β activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αβ fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for NaV1.1, their unusual dual mode of action provides an alternative approach to the development of selective NaV1.1 modulators for the treatment of DS.

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric β-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.

Voltage-gated sodium (NaV) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal NaV channels inhibiting peak current of hNaV1.1, rNaV1.2, hNaV1.6, and hNaV1.7 while concurrently inhibiting fast inactivation of hNaV1.1 and rNaV1.3. The DII and DIV S3-S4 loops of NaV channel voltage sensors are important for the interaction of Pre1a with NaV channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between NaV channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of NaV1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of NaV channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.

Stroke is the second-leading cause of death worldwide, yet there are no drugs available to protect the brain from stroke-induced neuronal injury. Acid-sensing ion channel 1a (ASIC1a) is the primary acid sensor in mammalian brain and a key mediator of acidosis-induced neuronal damage following cerebral ischemia. Genetic ablation and selective pharmacologic inhibition of ASIC1a reduces neuronal death following ischemic stroke in rodents. Here, we demonstrate that Hi1a, a disulfide-rich spider venom peptide, is highly neuroprotective in a focal model of ischemic stroke. Nuclear magnetic resonance structural studies reveal that Hi1a comprises two homologous inhibitor cystine knot domains separated by a short, structurally well-defined linker. In contrast with known ASIC1a inhibitors, Hi1a incompletely inhibits ASIC1a activation in a pH-independent and slowly reversible manner. Whole-cell, macropatch, and single-channel electrophysiological recordings indicate that Hi1a binds to and stabilizes the closed state of the channel, thereby impeding the transition into a conducting state. Intracerebroventricular administration to rats of a single small dose of Hi1a (2 ng/kg) up to 8 h after stroke induction by occlusion of the middle cerebral artery markedly reduced infarct size, and this correlated with improved neurological and motor function, as well as with preservation of neuronal architecture. Thus, Hi1a is a powerful pharmacological tool for probing the role of ASIC1a in acid-mediated neuronal injury and various neurological disorders, and a promising lead for the development of therapeutics to protect the brain from ischemic injury.

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (Na

This thesis describes the functional and structural characterisation of several novel peptides that modulate the activity of voltage-gated sodium channel 1.1 (NaV1.1), a putative target for the development of therapeutics to treat Dravet syndrome (DS) epilepsy.Voltage-gated sodium (NaV) channels serve a fundamental role in carrying electrical signals throughout the body. Inherited or acquired dysfunction of NaVchannels is associated with a number of human diseases, including epilepsy, cardiac arrhythmia, and chronic pain conditions. The NaV1.1 subtype, which is expressed in peripheral sensory neurons and fast-spiking GABAergic inhibitory interneurons in the brain, governs the proper excitation/inhibition balance in neuronal networks. Consequently, heterozygous loss-of-function mutations in SCN1A, the gene encoding the ion conducting α-subunit of the NaV1.1, lead to DS. DS is a rare, pharmacoresistant childhood epilepsy characterised by early-onset seizures, cognitive deficit, autistic-like behaviours, and premature death. The NaV1.1 haploinsufficiency in DS disturbs the normal balance of excitation and inhibition in the brain. We propose that this deficit could be ameliorated by a drug that selectively activatesthe smaller-than-normal population of functional NaV1.1 channels in DS interneurons, thus restoring their ability to regulate brain excitability. The overall aim of this project was to characterise novel NaV1.1-selective agonists in order to provide drug leads with potential for treatment of DS epilepsy.Designed by Nature as chemical weapons, arthropod venoms have proven to be an untapped source of ion channel modulators that can be repurposed as pharmacological tools for studying these large integral membrane proteins, and as leads for the development of new therapeutics. Recently, two disulfide-rich peptides (Hm1a and Hm1b) were isolated from the venom of the tarantula Heteroscodra maculata, and shown to specifically activate NaV1.1. A previous study demonstrated that intracerebroventricular (ICV) infusion of Hm1a restores the function of inhibitory interneurons in SCN1Aknockout mice by selectively activating NaV1.1 channels (Richards et al., 2018). However, somewhat surprisingly for a disulfide-rich venom peptide, Hm1a was poorly stable in cerebrospinal fluid (CSF) with a half-life of ~1.7 h. Given the limited amount of crude H. maculatavenom, Chapter Two describes the optimisation of recombinant production of correctly folded Hm1b (rHm1b). Despite the high degree of sequence similarity between Hm1a and Hm1b, rHm1b is much more stable than Hm1a in both human serum and human CSF (t1/2>40 h and >70 h, respectively). Furthermore, structural studies indicated that rHm1b exhibits an inhibitor cystine knot (ICK) motif. By comparing the molecular surface of Hm1a and rHm1b, we identified a conserved hydrophobic patch in these toxins that likely mediates their interaction with the NaV1.1 channel. Although in vivo studies have thus far focused on Hm1a, the easier production and higher in vivostability of rHm1b suggest that it might represent the better molecule to progress towards human clinical trials.Chapter Three explores the pharmacological profiles of native and recombinant versions of Hm1b on human NaV1.1–1.7 channel subtypes expressed in a mammalian-cell background using an automated whole-cell patch-clamp electrophysiology system. rHm1b is a highly potent and selective modulator of NaV1.1 and NaV1.3 in the low nanomolar range. However, rHm1b potentiates NaV1.3 tolesser extent than NaV1.1, and induces a smaller sustained current. Electrophysiology studies have shown that at any given potential within the physiologically relevant range, rHm1b promotes NaV1.1 activation at the resting membrane potential and inhibits fast inactivation, indicating that rHm1b stably traps the domain IV voltage sensors of NaV1.1 and NaV1.3 in the partially activated state, where the channels would maintain a persistent open state but not sufficient to trigger inactivation.In addition to the studies on rHm1b, Chapter Four describes the isolation and characterisation of two novel peptides (Hj1a and Hj1b) from venom of the scorpion Hottentotta jayakari. The primary structures of these two peptides were determined by analysis of a venom-gland transcriptome and proteomic data. Sequence alignments revealed a high degree of similarity to previously reported α-scorpion toxins. Recombinant peptides were produced, and their pharmacological profiles were characterised by electrophysiology measurements on human NaV1.1–1.7 channel subtypes. Hj1a and Hj1b were found to modulate NaV1.1 via a mechanism similar to that of classic gating-modifier toxins by enhancing channel activation and inhibiting fast inactivation. Both peptides also demonstrate diverse agonistic activity on NaV1.4–1.7 in the low nanomolar range. The original aims of this work were in part achieved, although the NaV1.1 agonists we found are not subtype-selective.Collectively, it is anticipated that future structure-activity relationship studies will facilitate rational engineering of peptide variants with improved potency and selectivity for NaV1.1.

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC50 = 160 pM). The native peptide also inhibits Kv1.1, Kv1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of Kv1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/β (CSα/β) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against Kv1.1–1.3 and Kv1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all Kv channels tested, without perturbing the toxin’s structure. Reciprocal mutations in the pore of Uro-sensitive Kv1.2 and Uro-resistant Kv1.5 channels revealed a direct interaction between Urotoxin and the Kv channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its Kv1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the Kv1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of Kv1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin.

Dravet syndrome (DS) is a catastrophic epileptic encephalopathy characterised by childhood-onset polymorphic seizures, multiple neuropsychiatric comorbidities, and increased risk of sudden death. Heterozygous loss-of-function mutations in one allele of SCN1A, the gene encoding the voltage-gated sodium channel 1.1 (NaV1.1), lead to DS. NaV1.1 is primarily found in the axon initial segment of fast-spiking GABAergic inhibitory interneurons in the brain, and the principle mechanism proposed to underlie seizure genesis in DS is loss of inhibitory input due to dysfunctional firing of GABAergic interneurons. We hypothesised that DS symptoms could be ameliorated by a drug that activates the reduced population of functional NaV1.1 channels in DS interneurons. We recently identified two homologous disulfide-rich spider-venom peptides (Hm1a and Hm1b) that selectively potentiate NaV1.1, and showed that selective activation of NaV1.1 by Hm1a restores the function of inhibitory interneurons in a mouse model of DS. Here we produced recombinant Hm1b (rHm1b) using an E. coli periplasmic expression system, and examined its selectivity against a panel of human NaV subtypes using whole-cell patch-clamp recordings. rHm1b is a potent and highly selective agonist of NaV1.1 and NaV1.3 (EC50 ~12 nM for both). rHm1b is a gating modifier that shifts the voltage dependence of channel activation and inactivation to hyperpolarised and depolarised potentials respectively, presumably by interacting with the channel’s voltage-sensor domains. Like Hm1a, the structure of rHm1b determined by using NMR revealed a classical inhibitor cystine knot (ICK) motif. However, we show that rHm1b is an order of magnitude more stable than Hm1a in human cerebrospinal fluid. Overall, our data suggest that rHm1b is an exciting lead for a precision therapeutic targeted against DS.

Spiders are the most successful venomous animals on the planet, with more than 47,000 extant species. Most spider venoms are dominated by disulfide-rich peptides (DRPs) with a diverse range of pharmacological activities. Although some venoms contain thousands of unique peptides, little is known about the mechanisms used to generate such complex chemical arsenals. We used a combined transcriptomic, proteomic and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins, more than described for any other arachnid. We show that 15 of the 26 DRP superfamilies form an ultra-stable inhibitor cystine knot motif, and that these DRPs are the major contributor to the diversity of the venom peptidome. NMR data reveal that most of these DRPs are structurally related and range in complexity from simple to highly elaborated knottin domains that likely evolved from a single ancestral fold.

Structures of voltage-gated sodium channels In “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Na v ) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Na v channel, and Shen et al. report the structures of an insect Na v channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery. Science , this issue p. eaau2486 , p. eaau2596

Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide &omega, HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.

Recently, we and other groups revealed that gain-of-function mutations in the human ether a go-go voltage-gated potassium channel hEAG1 (Kv10.1) lead to developmental disorders with associated infantile-onset epilepsy. However, the physiological role of hEAG1 in the central nervous system remains elusive. Potent and selective antagonists of hEAG1 are therefore much sought after, both as pharmacological tools for studying the (patho)physiological functions of this enigmatic channel and as potential leads for development of anti-epileptic drugs. Since animal venoms are a rich source of potent ion channel modifiers that have been finely tuned by millions of year of evolution, we screened 108 arachnid venoms for hEAG1 inhibitors using electrophysiology. Two hit peptides (Aa1a and Ap1a) were isolated, sequenced, and chemically synthesised for structure-function studies. Both of these hEAG1 inhibitors are C-terminally amidated peptides containing an inhibitor cystine knot motif, which provides them with exceptional stability in both plasma and cerebrospinal fluid. Aa1a and Ap1a are the most potent peptidic inhibitors of hEAG1 reported to date, and they present a novel mode of action by targeting both the activation and inactivation gating of the channel. These peptides should be useful pharmacological tools for probing hEAG1 function as well as informative leads for the development of novel anti-epileptic drugs.

Many spider-venom peptides are known to modulate the activity of the voltage-gated sodium (NaV) subtype 1.7 (NaV1.7) channel, which has emerged as a promising analgesic target. In particular, a class of spider-venom peptides (NaSpTx1) has been found to potently inhibit NaV1.7 (nanomolar IC50), and has been shown to produce analgesic effects in animals. However, one member of this family [µ-TRTX-Hhn2b (Hhn2b)] does not inhibit mammalian NaV channels expressed in dorsal root ganglia at concentrations up to 100 µM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b, leading us to identify two nonpharmacophore residues that contribute to human NaV1.7 (hNaV1.7) inhibition by nonoverlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNaV1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild-type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.

Bacterial division begins with the formation of a contractile protein ring at midcell, which constricts the bacterial envelope to generate two daughter cells. The central component of the division ring is FtsZ, a tubulin-like protein capable of self-assembling into filaments which further associate into a higher order structure known as the Z ring. Proteins that bind to FtsZ play a crucial role in the formation and regulation of the Z ring. One such protein is ZapA, a widely conserved 21 kDa homodimeric protein that associates with FtsZ filaments and promotes their bundling. Although ZapA was discovered more than a decade ago, the structural details of its interaction with FtsZ remain unknown. In this work, backbone and side chain NMR assignments for the Geobacillus stearothermophilus ZapA homodimer are described. We titrated FtsZ into (15)N(2)H-ZapA and mapped ZapA residues whose resonances are perturbed upon FtsZ binding. This information provides a structural understanding of the interaction between FtsZ and ZapA.

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Remerciements :- Dr. Jacques Delabie- Andrea Yockey-Dejean- Geoff Brown- Jean-Pierre Andrieu; Background: Most ant venoms consist predominantly of small linear peptides, although some contain disulfide linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides.Methods: The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U-1-PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels.Results: Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal beta-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (Ca(v)1).Conclusions: Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms.General significance: This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides.

We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. The reduced structure shows a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helical domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerably more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike's information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the pico- to nanosecond and micro- to millisecond timescale. Many significant changes in dynamics are located either close to the active site or at the insertion points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect the catalytic cleft of the enzyme. Taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.

The α1β1 integrin receptor binds to its main extracellular ligand, collagen, through an inserted domain in its α-subunit called the αI domain (αI). αI contains a metal binding site that allows collagen to coordinate to the domain through a divalent metal ion. Here we report the backbone assignments of the apo and Mg(2+) bound state of the isolated human α1I and the chemical shift changes resulting from metal coordination.

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.