Proteomic Investigation of Cape Cobra (Naja nivea) Venom Reveals First Evidence of Quaternary Protein Structures.

Naja nivea (N. nivea) is classed as a category one snake by the World Health Organization since its envenomation causes high levels of mortality and disability annually. Despite this, there has been little research into the venom composition of N. nivea, with only one full venom proteome published to date. Our current study separated N. nivea venom using size exclusion chromatography before utilizing a traditional bottom-up proteomics approach to unravel the composition of the venom proteome. As expected by its clinical presentation, N. nivea venom was found to consist mainly of neurotoxins, with three-finger toxins (3FTx), making up 76.01% of the total venom proteome. Additionally, cysteine-rich secretory proteins (CRISPs), vespryns (VESPs), cobra venom factors (CVFs), 5'-nucleotidases (5'NUCs), nerve growth factors (NGFs), phospholipase A2s (PLA2), acetylcholinesterases (AChEs), Kunitz-type serine protease inhibitor (KUN), phosphodiesterases (PDEs), L-amino acid oxidases (LAAOs), hydrolases (HYDs), snake venom metalloproteinases (SVMPs), and snake venom serine protease (SVSP) toxins were also identified in decreasing order of abundance. Interestingly, contrary to previous reports, we find PLA2 toxins in N. nivea venom. This highlights the importance of repeatedly profiling the venom of the same species to account for intra-species variation. Additionally, we report the first evidence of covalent protein complexes in N. nivea venom, which likely contribute to the potency of this venom.

Native Mass Spectrometric Insights into the Formation and Stability of DNA Triplexes.

Deoxyribonucleic acid is a genetic biomacromolecule that contains the inherited information required to build and maintain a living organism. While the canonical duplex DNA structure is rigorously characterized, the structure and function of higher order DNA structures such as DNA triplexes are comparatively poorly understood. Previous literature has shown that these triplexes can form under physiological conditions, and native mass spectrometry offers a useful platform to study their formation and stability. However, experimental conditions including buffer salt concentration, pH, and instrumentation parameters such as ion mode can confound analysis by impacting the amount of triplex observed by mass spectrometry. Using model 30mer Y-type triplex sequences, we demonstrate the influence a range of experimental variables have on the detection of DNA triplex structures, informing suitable conditions for the study. When carefully considered conditions are used, mass spectrometry offers a powerful complementary tool for the analysis of higher order DNA assemblies.

The Eastern Bandy Bandy Vermicella annulata, expresses high abundance of SVMP, CRiSP and Kunitz protein families in its venom proteome.

The Australian elapid snake radiation (Hydrophiinae) has evolved in the absence of competition from other advanced snakes. This has resulted in ecological specialisation in Australian elapids and the potential for venom proteomes divergent to other elapids. We characterised the venom of the Australian elapid Vermicella annulata (eastern bandy bandy). The venom was analysed using a two-dimensional fractionation process consisting of reverse-phase high-performance liquid chromatography then sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by bottom-up proteomics. Resulting peptides were matched to a species-specific transcriptome and 87% of the venom was characterised. We identified 11 toxins in the venom from six families: snake venom metalloproteinases (SVMP; 24.2%; two toxins) that are class P-III SVMPs containing a disintegrin-like domain, three-finger toxins (3FTx; 21.6%; five toxins), kunitz peptides (KUN; 19.5%; one toxin), cysteine-rich secretory proteins (CRiSP; 18%; one toxin), and phospholipase A2 (PLA2; 4%; two toxins). The venom had low toxin diversity with five protein families having one or two toxins, except for 3FTx with five different toxins. V. annulata expresses an unusual venom proteome, with high abundances of CRiSP, KUN and SVMP, which are not normally highly expressed in elapid venoms. This unusual venom composition could be an adaptation to its specialised diet. BIOLOGICAL SIGNIFICANCE: Although the Australian elapid radiation represents the most extensive speciation event of elapids on any continent, with 100 terrestrial species, the venom composition of these snakes has rarely been investigated, with only five species currently characterised. Here we provide the venom proteome of a sixth species, Vermicella annulata. The venom of this species could be particularly informative from an evolutionary perspective, as it is an extreme dietary specialist, only preying on blind snakes (Typhlopidae). We show that V. annulata expresses a highly unusual venom for an elapid, due to the high abundance of the protein families SVMP, CRiSP, and KUN, which together make up 61% of the venom. When averaged across all species, a typical elapid venom is 82% PLA2 and 3FTx. This is the second recorded instance of an Australian elapid having evolved highly divergent venom expression.

Characterisation of the forest cobra (Naja melanoleuca) venom using a multifaceted mass spectrometric-based approach.

Snake venoms consist of highly biologically active proteins and peptides that are responsible for the lethal physiological effects of snakebite envenomation. In order to guide the development of targeted antivenom strategies, comprehensive understanding of venom compositions and in-depth characterisation of various proteoforms, often not captured by traditional bottom-up proteomic workflows, is necessary. Here, we employ an integrated 'omics' and intact mass spectrometry (MS)-based approach to profile the heterogeneity within the venom of the forest cobra (Naja melanoleuca), adopting different analytical strategies to accommodate for the dynamic molecular mass range of venom proteins present. The venom proteome of N. melanoleuca was catalogued using a venom gland transcriptome-guided bottom-up proteomics approach, revealing a venom consisting of six toxin superfamilies. The subtle diversity present in the venom components was further explored using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to intact MS. This approach showed a significant increase in the number of venom proteoforms within various toxin families that were not captured in previous studies. Furthermore, we probed at the higher-order structures of the larger venom proteins using a combination of native MS and mass photometry and revealed significant structural heterogeneity along with extensive post-translational modifications in the form of glycosylation in these larger toxins. Here, we show the diverse structural heterogeneity of snake venom proteins in the venom of N. melanoleuca using an integrated workflow that incorporates analytical strategies that profile snake venom at the proteoform level, complementing traditional venom characterisation approaches.

Efficient biocatalytic C-H bond oxidation: an engineered heme-thiolate peroxygenase from a thermostable cytochrome P450.

A highly sought after reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds. We demonstrate the hydroxylation of fatty acids using an engineered thermostable archaeal cytochrome P450 enzyme. By replacing a seven amino acid section of the I-helix, the nicotinamide cofactor-dependent monooxygenase was converted into a hydrogen peroxide using peroxygenase, enabling the efficient biocatalytic oxidation of C-H bonds at room temperature to 90 °C.

An approach to quantify ortho-phthalaldehyde contamination on work surfaces.

Ortho-phthalaldehyde (OPA) is used as a high-level disinfectant for reusable medical devices in healthcare settings. The ACGIH recently adopted a Threshold Limit Value-Surface Limit (TLV-SL; 25 µg/100 cm2) for OPA surface contamination to prevent induction of dermal and respiratory sensitization following dermal exposure. However, there is no current validated method to measure OPA surface contamination. This study aimed to develop a standardized approach for sample collection and quantitative determination of OPA from work surfaces for use in risk assessment practices. The reported method utilises readily available commercial wipes to collect surface samples coupled with direct detection of OPA via liquid chromatography time of flight mass spectrometry (LC-ToF-MS). This approach avoided complex derivatization steps commonly required for the analysis of aldehydes. Method evaluation was conducted in accordance with the Occupational Safety and Health Administration (OSHA) surface sampling guidelines. Overall recoveries of 25 µg/100 cm2 of OPA from stainless steel and glass surfaces were 70% and 72%, respectively. The reported LOD for this method was 1.1 µg/sample and the LOQ was 3.7 µg/sample. OPA remained stable on the sampling medium for up to 10 days, when stored at 4 °C. The method was demonstrated in a workplace surface assessment at a local hospital sterilising unit, successfully detecting OPA on work surfaces. This method is intended to supplement airborne exposure assessment and provide a quantitative assessment tool for potential dermal exposure. When used in conjunction with a thorough occupational hygiene program that includes hazard communication, engineering controls, and personal protective equipment, skin exposure and consequent sensitization risks in the workplace can be minimized.

Better late than never: Optimising the proteomic analysis of field-collected octopus.

Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10% additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

Structural Analysis and Identity Confirmation of Anthocyanins in Brassica oleracea Extracts by Direct Injection Ion Mobility-Mass Spectrometry.

Anthocyanins are a subclass of plant-derived flavonoids that demonstrate immense structural heterogeneity which is challenging to capture in complex extracts by traditional liquid chromatography-mass spectrometry (MS)-based approaches. Here, we investigate direct injection ion mobility-MS as a rapid analytical tool to characterize anthocyanin structural features in red cabbage (Brassica oleracea) extracts. Within a 1.5 min sample run time, we observe localization of structurally similar anthocyanins and their isobars into discrete drift time regions based upon their degree of chemical modifications. Furthermore, drift time-aligned fragmentation enables simultaneous collection of MS, MS/MS, and collisional cross-section data for individual anthocyanin species down to a low picomole scale to generate structural identifiers for rapid identity confirmation. We finally identify anthocyanins in three other Brassica oleracea extracts based on red cabbage anthocyanin identifiers to demonstrate our high-throughput approach. Direct injection ion mobility-MS therefore provides wholistic structural information on structurally similar, and even isobaric, anthocyanins in complex plant extracts, which can inform the nutritional value of a plant and bolster drug discovery pipelines.

PPARγ Corepression Involves Alternate Ligand Conformation and Inflation of H12 Ensembles.

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

Engineering C-C Bond Cleavage Activity into a P450 Monooxygenase Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

Escherichia coli YgiC and YjfC Possess Peptide─Spermidine Ligase Activity.

Polyamines and polyamine-containing metabolites are involved in many cellular processes related to bacterial cell growth and survival. In Escherichia coli, the bifunctional enzyme glutathionylspermidine synthetase/amidase (GspSA) controls the production of glutathionylspermidine, which has a protective role against oxidative stress. E. coli also encodes two enzymes with homology to the synthetase domain of GspSA, YgiC, and YjfC; however, these do not catalyze the formation of glutathionylspermidine, and their catalytic function remained unknown. Here, we detail the structural and functional characterization of YgiC and YjfC. Using X-ray crystallography, the high-resolution crystal structures of YgiC and YjfC were obtained. This revealed that YgiC and YjfC possess multiple substitutions in key residues required for binding of glutathione in GspSA. Despite this difference, these enzymes share a similar active site structure to GspSA, suggesting that they catalyze the formation of an alternate peptide─spermidine conjugate. As the physiological substrates of YgiC and YjfC are unknown, this was probed using the peptide triglycine as a model substrate. A combination of enzyme activity assays and mass spectrometry revealed that YgiC and YjfC can function as peptide-spermidine ligases, forming a triglycine-spermidine conjugate. For both enzymes, conjugate formation was only observed in the presence of spermidine, but not other common polyamines, supporting that spermidine or a spermidine derivative is the physiological substrate. Importantly, since YgiC and YjfC are widely distributed in Gram-negative bacterial species, this suggests that these enzymes function in a conserved cellular process, representing a currently unknown aspect of bacterial polyamine metabolism.

Mass spectrometric insights into protein aggregation.

Protein aggregation is now recognized as a generic and significant component of the protein energy landscape. Occurring through a complex and dynamic pathway of structural interconversion, the assembly of misfolded proteins to form soluble oligomers and insoluble aggregates remains a challenging topic of study, both in vitro and in vivo. Since the etiology of numerous human diseases has been associated with protein aggregation, and it has become a field of increasing importance in the biopharmaceutical industry, the biophysical characterization of protein misfolded states and their aggregation mechanisms continues to receive increased attention. Mass spectrometry (MS) has firmly established itself as a powerful analytical tool capable of both detection and characterization of proteins at all levels of structure. Given inherent advantages of biological MS, including high sensitivity, rapid timescales of analysis, and the ability to distinguish individual components from complex mixtures with unrivalled specificity, it has found widespread use in the study of protein aggregation, importantly, where traditional structural biology approaches are often not amenable. The present review aims to provide a brief overview of selected MS-based approaches that can provide a range of biophysical descriptors associated with protein conformation and the aggregation pathway. Recent examples highlight where this technology has provided unique structural and mechanistic understanding of protein aggregation.

Salivary inflammatory biomarkers are predictive of mild cognitive impairment and Alzheimer's disease in a feasibility study.

Alzheimer's disease (AD) is an insidious disease. Its distinctive pathology forms over a considerable length of time without symptoms. There is a need to detect this disease, before even subtle changes occur in cognition. Hallmark AD biomarkers, tau and amyloid-ß, have shown promising results in CSF and blood. However, detecting early changes in these biomarkers and others will involve screening a wide group of healthy, asymptomatic individuals. Saliva is a feasible alternative. Sample collection is economical, non-invasive and saliva is an abundant source of proteins including tau and amyloid-ß. This work sought to extend an earlier promising untargeted mass spectrometry study in saliva from individuals with mild cognitive impairment (MCI) or AD with age- and gender-matched cognitively normal from the South Australian Neurodegenerative Disease cohort. Five proteins, with key roles in inflammation, were chosen from this study and measured by ELISA from individuals with AD (n = 16), MCI (n = 15) and cognitively normal (n = 29). The concentrations of Cystatin-C, Interleukin-1 receptor antagonist, Stratifin, Matrix metalloproteinase 9 and Haptoglobin proteins had altered abundance in saliva from AD and MCI, consistent with the earlier study. Receiver operating characteristic analysis showed that combinations of these proteins demonstrated excellent diagnostic accuracy for distinguishing both MCI (area under curve = 0.97) and AD (area under curve = 0.97) from cognitively normal. These results provide evidence for saliva being a valuable source of biomarkers for early detection of cognitive impairment in individuals on the AD continuum and potentially other neurodegenerative diseases.

Preliminary Development and Testing of C595 Radioimmunoconjugates for Targeting MUC1 Cancer Epitopes in Pancreatic Ductal Adenocarcinoma.

Mucin 1 is a transmembrane glycoprotein which overexpresses cancer-specific epitopes (MUC1-CE) on pancreatic ductal adenocarcinoma (PDAC) cells. As PDAC is a low survival and highly aggressive malignancy, developing radioimmunoconjugates capable of targeting MUC1-CE could lead to improvements in PDAC outcomes. The aim of this study was to develop and perform preliminary testing of diagnostic and therapeutic radioimmunoconjugates for PDAC using an anti-MUC1 antibody, C595. Firstly, p-SCN-Bn-DOTA was conjugated to the C595 antibody to form a DOTA-C595 immunoconjugate. The stability and binding affinity of the DOTA-C595 conjugate was evaluated using mass spectrometry and ELISA. DOTA-C595 was radiolabelled to Copper-64, Lutetium-177, Gallium-68 and Technetium-99m to form novel radioimmunoconjugates. Cell binding assays were performed in PANC-1 (strong MUC1-CE expression) and AsPC-1 (weak MUC1-CE expression) cell lines using 64Cu-DOTA-C595 and 177Lu-DOTA-C595. An optimal molar ratio of 4:1 DOTA groups per C595 molecule was obtained from the conjugation process. DOTA-C595 labelled to Copper-64, Lutetium-177, and Technetium-99m with high efficiency, although the Gallium-68 labelling was low. 177Lu-DOTA-C595 demonstrated high cellular binding to the PANC-1 cell lines which was significantly greater than AsPC-1 binding at concentrations exceeding 100 nM (p < 0.05). 64Cu-DOTA-C595 showed similar binding to the PANC-1 and AsPC-1 cells with no significant differences observed between cell lines (p > 0.05). The high cellular binding of 177Lu-DOTA-C595 to MUC1-CE positive cell lines suggests promise as a therapeutic radioimmunoconjugate against PDAC while further work is required to harness the potential of 64Cu-DOTA-C595 as a diagnostic radioimmunoconjugate.

Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism.

Enzymes involved in Staphylococcus aureus amino acid metabolism have recently gained traction as promising targets for the development of new antibiotics, however, not all aspects of this process are understood. The ATP-grasp superfamily includes enzymes that predominantly catalyze the ATP-dependent ligation of various carboxylate and amine substrates. One subset, ʟ-amino acid ligases (LALs), primarily catalyze the formation of dipeptide products in Gram-positive bacteria, however, their involvement in S. aureus amino acid metabolism has not been investigated. Here, we present the characterization of the putative ATP-grasp enzyme (SAOUHSC_02373) from S. aureus NCTC 8325 and its identification as a novel LAL. First, we interrogated the activity of SAOUHSC_02373 against a panel of ʟ-amino acid substrates. As a result, we identified SAOUHSC_02373 as an LAL with high selectivity for ʟ-aspartate and ʟ-methionine substrates, specifically forming an ʟ-aspartyl-ʟ-methionine dipeptide. Thus, we propose that SAOUHSC_02373 be assigned as ʟ-aspartate-ʟ-methionine ligase (LdmS). To further understand this unique activity, we investigated the mechanism of LdmS by X-ray crystallography, molecular modeling, and site-directed mutagenesis. Our results suggest that LdmS shares a similar mechanism to other ATP-grasp enzymes but possesses a distinctive active site architecture that confers selectivity for the ʟ-Asp and ʟ-Met substrates. Phylogenetic analysis revealed LdmS homologs are highly conserved in Staphylococcus and closely related Gram-positive Firmicutes. Subsequent genetic analysis upstream of the ldmS operon revealed several trans-acting regulatory elements associated with control of Met and Cys metabolism. Together, these findings support a role for LdmS in Staphylococcal sulfur amino acid metabolism.

Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity.

The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson's disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36-42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77-82 NAC) and heptapeptide (GVLYVGS-Aib, 36-42 NTR) containing a self-recognition component unique to αS, as well as a C-terminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregation-prone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson's disease.

Polyphenol characterisation and diverse bioactivities of native Australian lilly pilly (Syzygium paniculatum) extract.

Magenta lilly pilly (Syzygium paniculatum) is an Australian native tree that produces berry fruits that are rich in phytochemicals reportedly beneficial to human health. Here we explored the biological activities of polyphenol-enriched extracts from the magenta lilly pilly fruit, benchmarking it against traditional sources including purple sweet potato and blackberry. We show that the extracts exert potent antioxidant and neuroprotective properties as well as antimicrobial activity against Staphylococcus aureus. The phenolic composition of lilly pilly was investigated using liquid chromatography coupled to mass spectrometry (HPLC-DAD-MS), revealing anthocyanins to be the primary component in high abundance compared to traditional anthocyanin-containing plants. Three anthocyanins from lilly pilly, along with their glycosylation patterns and stability, were characterised. Altogether, our results demonstrate the potential to exploit magenta lilly pilly fruits as a high-yielding source of phenolics with beneficial biological properties of potential interest for multiple downstream applications.

The Unusual Metalloprotease-Rich Venom Proteome of the Australian Elapid Snake Hoplocephalus stephensii.

The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents; however, their venom compositions are poorly known. The Australian elapid Hoplocephalus stephensii (Stephen's banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of H. stephensii. 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP; 36.7%; three identified toxins), phospholipase A2 (PLA2; 24.0%; five identified toxins), three-finger toxin (3FTx; 10.2%; two toxins) and snake venom serine protease (SVSP; 5.9%; one toxin; Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO; 10.8%; one toxin), natriuretic peptide (NP; 0.8%; two toxins), cysteine-rich secretory protein (CRiSP; 1.7%; two toxins), c-type lectin (CTL; 1.1%; one toxin), and one minor protein family, nerve growth factor (NGF; 0.8%; one toxin). The venom composition of H. stephensii differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. H. stephensii venom appeared to have less toxin diversity than other elapids, with only 18 toxins making up three-quarters of the venom.

A structural model of the human plasminogen and Aspergillus fumigatus enolase complex.

The metabolic enzyme, enolase, plays a crucial role in the cytoplasm where it maintains cellular energy production within the process of glycolysis. The main role of enolase in glycolysis is to convert 2-phosphoglycerate to phosphoenolpyruvate; however, enolase can fulfill roles that deviate from this function. In pathogenic bacteria and fungi, enolase is also located on the cell surface where it functions as a virulence factor. Surface-expressed enolase is a receptor for human plasma proteins, including plasminogen, and this interaction facilitates nutrient acquisition and tissue invasion. A novel approach to developing antifungal drugs is to inhibit the formation of this complex. To better understand the structure of enolase and the interactions that may govern complex formation, we have solved the first X-ray crystal structure of enolase from Aspergillus fumigatus (2.0 Å) and have shown that it preferentially adopts a dimeric quaternary structure using native mass spectrometry. Two additional X-ray crystal structures of A. fumigatus enolase bound to the endogenous substrate 2-phosphoglycerate and product phosphoenolpyruvate were determined and kinetic characterization was carried out to better understand the details of its canonical function. From these data, we have produced a model of the A. fumigatus enolase and human plasminogen complex to provide structural insights into the mechanisms of virulence and aid future development of small molecules or peptidomimetics for antifungal drug design.

Structural insights into the antifungal drug target guanosine monophosphate synthase from Aspergillus fumigatus.

Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Šresolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC50 = 16.6 ± 2.4 µM) and 6-diazo-5-oxo-L-norleucine (IC50 = 29.6 ± 5.6 µM) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug-discovery endeavours.