Biochemistry and Pharmacology - Research Publications

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    CSM-Potential: mapping protein interactions and biological ligands in 3D space using geometric deep learning
    Rodrigues, CHM ; Ascher, DB (OXFORD UNIV PRESS, 2022-05-24)
    Recent advances in protein structural modelling have enabled the accurate prediction of the holo 3D structures of almost any protein, however protein function is intrinsically linked to the interactions it makes. While a number of computational approaches have been proposed to explore potential biological interactions, they have been limited to specific interactions, and have not been readily accessible for non-experts or use in bioinformatics pipelines. Here we present CSM-Potential, a geometric deep learning approach to identify regions of a protein surface that are likely to mediate protein-protein and protein-ligand interactions in order to provide a link between 3D structure and biological function. Our method has shown robust performance, outperforming existing methods for both predictive tasks. By assessing the performance of CSM-Potential on independent blind tests, we show that our method was able to achieve ROC AUC values of up to 0.81 for the identification of potential protein-protein binding sites, and up to 0.96 accuracy on biological ligand classification. Our method is freely available as a user-friendly and easy-to-use web server and API at http://biosig.unimelb.edu.au/csm_potential.
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    Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen
    Gorrie, CL ; Mirceta, M ; Wick, RR ; Judd, LM ; Lam, MMC ; Gomi, R ; Abbott, IJ ; Thomson, NR ; Strugnell, RA ; Pratt, NF ; Garlick, JS ; Watson, KM ; Hunter, PC ; Pilcher, DV ; McGloughlin, SA ; Spelman, DW ; Wyres, KL ; Jenney, AWJ ; Holt, KE (NATURE PORTFOLIO, 2022-05-31)
    Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.
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    Editorial: Venoms and Toxins: Functional Omics and Pharmacological Insights.
    Tan, CH ; Tan, KY ; Jackson, TNW (Frontiers Media SA, 2022)
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    Emerging COVID-19 coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26
    Vankadari, N ; Wilce, JA (TAYLOR & FRANCIS LTD, 2020-01-01)
    The recent outbreak of pneumonia-causing COVID-19 in China is an urgent global public health issue with an increase in mortality and morbidity. Here we report our modelled homo-trimer structure of COVID-19 spike glycoprotein in both closed (ligand-free) and open (ligand-bound) conformation, which is involved in host cell adhesion. We also predict the unique N- and O-linked glycosylation sites of spike glycoprotein that distinguish it from the SARS and underlines shielding and camouflage of COVID-19 from the host the defence system. Furthermore, our study also highlights the key finding that the S1 domain of COVID-19 spike glycoprotein potentially interacts with the human CD26, a key immunoregulatory factor for hijacking and virulence. These findings accentuate the unique features of COVID-19 and assist in the development of new therapeutics.
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    The first reptilian allergen and major allergen for fish-allergic patients: Crocodile beta-parvalbumin
    Ruethers, T ; Nugraha, R ; Taki, AC ; O'Malley, A ; Karnaneedi, S ; Zhang, S ; Kapingidza, AB ; Mehr, S ; Kamath, SD ; Chruszcz, M ; Mackay, G ; Campbell, DE ; Lopata, AL (WILEY, 2022-05-01)
    BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile β-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for β-PV and epitopes predicted, explaining frequent IgE-cross-binding of β-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (β-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.
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    Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity
    Lu, J ; Azad, MAK ; Moreau, JLM ; Zhu, Y ; Jiang, X ; Tonta, M ; Lam, R ; Wickremasinghe, H ; Zhao, J ; Wang, J ; Coleman, HA ; Formosa, LE ; Velkov, T ; Parkington, HC ; Combes, AN ; Rosenbluh, J ; Li, J (SPRINGER BASEL AG, 2022-06-01)
    Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.
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    Kaptive 2.0: updated capsule and lipopolysaccharide locus typing for the Klebsiella pneumoniae species complex
    Lam, MMC ; Wick, RR ; Judd, LM ; Holt, KE ; Wyres, KL (MICROBIOLOGY SOC, 2022-03-01)
    The outer polysaccharide capsule and lipopolysaccharide (LPS) antigens are key targets for novel control strategies targeting Klebsiella pneumoniae and related taxa from the K. pneumoniae species complex (KpSC), including vaccines, phage and monoclonal antibody therapies. Given the importance and growing interest in these highly diverse surface antigens, we had previously developed Kaptive, a tool for rapidly identifying and typing capsule (K) and outer LPS (O) loci from whole genome sequence data. Here, we report two significant updates, now freely available in Kaptive 2.0 (https://github.com/katholt/kaptive): (i) the addition of 16 novel K locus sequences to the K locus reference database following an extensive search of >17 000 KpSC genomes; and (ii) enhanced O locus typing to enable prediction of the clinically relevant O2 antigen (sub)types, for which the genetic determinants have been recently described. We applied Kaptive 2.0 to a curated dataset of >12 000 public KpSC genomes to explore for the first time, to the best of our knowledge, the distribution of predicted O (sub)types across species, sampling niches and clones, which highlighted key differences in the distributions that warrant further investigation. As the uptake of genomic surveillance approaches continues to expand globally, the application of Kaptive 2.0 will generate novel insights essential for the design of effective KpSC control strategies.
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    Mitochondrial COA7 is a heme-binding protein with disulfide reductase activity, which acts in the early stages of complex IV assembly
    Formosa, LE ; Maghool, S ; Sharpe, AJ ; Reljic, B ; Muellner-Wong, L ; Stroud, DA ; Ryan, MT ; Maher, MJ (NATL ACAD SCIENCES, 2022-02-25)
    Cytochrome c oxidase (COX) assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia, and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here, we show that loss of COA7 blocks complex IV assembly after the initial step where the COX1 module is built, progression from which requires the incorporation of copper and addition of the COX2 and COX3 modules. The crystal structure of COA7, determined to 2.4 Å resolution, reveals a banana-shaped molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds. COA7 interacts transiently with the copper metallochaperones SCO1 and SCO2 and catalyzes the reduction of disulfide bonds within these proteins, which are crucial for copper relay to COX2. COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. We therefore propose that COA7 is a heme-binding disulfide reductase for regenerating the copper relay system that underpins complex IV assembly.
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    cropCSM: designing safe and potent herbicides with graph-based signatures
    Pires, DE ; Stubbs, KA ; Mylne, JS ; Ascher, DB (OXFORD UNIV PRESS, 2022-02-24)
    Herbicides have revolutionised weed management, increased crop yields and improved profitability allowing for an increase in worldwide food security. Their widespread use, however, has also led to a rise in resistance and concerns about their environmental impact. Despite the need for potent and safe herbicidal molecules, no herbicide with a new mode of action has reached the market in 30 years. Although development of computational approaches has proven invaluable to guide rational drug discovery pipelines, leading to higher hit rates and lower attrition due to poor toxicity, little has been done in contrast for herbicide design. To fill this gap, we have developed cropCSM, a computational platform to help identify new, potent, nontoxic and environmentally safe herbicides. By using a knowledge-based approach, we identified physicochemical properties and substructures enriched in safe herbicides. By representing the small molecules as a graph, we leveraged these insights to guide the development of predictive models trained and tested on the largest collected data set of molecules with experimentally characterised herbicidal profiles to date (over 4500 compounds). In addition, we developed six new environmental and human toxicity predictors, spanning five different species to assist in molecule prioritisation. cropCSM was able to correctly identify 97% of herbicides currently available commercially, while predicting toxicity profiles with accuracies of up to 92%. We believe cropCSM will be an essential tool for the enrichment of screening libraries and to guide the development of potent and safe herbicides. We have made the method freely available through a user-friendly webserver at http://biosig.unimelb.edu.au/crop_csm.
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    Systematic evaluation of computational tools to predict the effects of mutations on protein stability in the absence of experimental structures
    Pan, Q ; Nguyen, TB ; Ascher, DB ; Pires, DE (OXFORD UNIV PRESS, 2022-03-10)
    Changes in protein sequence can have dramatic effects on how proteins fold, their stability and dynamics. Over the last 20 years, pioneering methods have been developed to try to estimate the effects of missense mutations on protein stability, leveraging growing availability of protein 3D structures. These, however, have been developed and validated using experimentally derived structures and biophysical measurements. A large proportion of protein structures remain to be experimentally elucidated and, while many studies have based their conclusions on predictions made using homology models, there has been no systematic evaluation of the reliability of these tools in the absence of experimental structural data. We have, therefore, systematically investigated the performance and robustness of ten widely used structural methods when presented with homology models built using templates at a range of sequence identity levels (from 15% to 95%) and contrasted performance with sequence-based tools, as a baseline. We found there is indeed performance deterioration on homology models built using templates with sequence identity below 40%, where sequence-based tools might become preferable. This was most marked for mutations in solvent exposed residues and stabilizing mutations. As structure prediction tools improve, the reliability of these predictors is expected to follow, however we strongly suggest that these factors should be taken into consideration when interpreting results from structure-based predictors of mutation effects on protein stability.