ORCID Profile
0000-0003-2420-4262
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Proteins and Peptides | Structural Biology (incl. Macromolecular Modelling) | Medicinal and Biomolecular Chemistry | Physical Chemistry (Incl. Structural) | Structural Chemistry and Spectroscopy | Characterisation of biological macromolecules | Characterisation of Biological Macromolecules | Biochemistry and Cell Biology | Structure and dynamics of materials | Biologically Active Molecules | Chemical Characterisation of Materials | Biochemistry and Cell Biology not elsewhere classified | Macromolecular and materials chemistry | Synthetic Biology | Electrochemical energy storage and conversion
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Physical Sciences |
Related Links
Publications
Classification of the human phox homology (PX) domains based on their phosphoinositide binding specificities
Publisher: Springer Science and Business Media LLC
Date: 04-04-2019
DOI: 10.1038/S41467-019-09355-Y
Abstract: Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3 P , others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3 P , non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3 P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.
Optimizing the transformation of HYSCORE data using the maximum entropy algorithm
Publisher: Elsevier BV
Date: 04-2019
DOI: 10.1016/J.JMR.2019.02.002
Abstract: Non-uniform s ling (NUS) in combination with the Maximum Entropy (MaxEnt) algorithm as applied to multi-dimensional NMR data has been thoroughly investigated and the NUS approach shown to provide significant sensitivity improvements as compared to methods using uniformly s led (US) data and the discrete Fourier transform (DFT). Hyperfine sublevel correlation (HYSCORE) is a standard pulse EPR experiment that can potentially benefit greatly from this approach, but the data present unique challenges as compared to NMR. HYSCORE data typically exhibit a very large range of peak intensities, signals are in the form of irregularly shaped ridges with variable intensities, and time traces are generally truncated to save measurement time. MaxEnt has the advantageous properties that it does not require US data, d ens weak signals (noise) and does not suffer from windowing artifacts due to truncation of the time traces. Critical to the success of the MaxEnt algorithm is the choice of the two input parameters aim and def which describe the data noise and contribution of entropy in the optimization, respectively. In this paper we expand our preliminary study on the application of MaxEnt to the reconstruction of HYSCORE spectra to include a detailed analysis on sensitivity to detect weak peaks, investigate the non-linearity of the transformation and ascertain if it can be characterized by the introduction of synthetic peaks, and define a general range for the choice of aim and def. Furthermore, the ability of the MaxEnt method to remove windowing artefacts in uniformly s led truncated HYSCORE data is described.
Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke schistosoma mansoni
Publisher: Elsevier BV
Date: 03-2014
Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain
Publisher: Springer Science and Business Media LLC
Date: 30-01-2014
DOI: 10.1038/NCOMMS4165
Abstract: Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general.
Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling
Publisher: Springer Science and Business Media LLC
Date: 31-07-2017
DOI: 10.1038/NSMB.3444
Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR
Publisher: Copernicus GmbH
Date: 24-02-2023
DOI: 10.5194/MR-4-57-2023
Abstract: Abstract. Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (atb.uq.edu.au, last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of side chains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein–protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.
Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function
Publisher: Springer Science and Business Media LLC
Date: 28-04-2023
DOI: 10.1038/S41467-023-37963-2
Abstract: Voltage-gated sodium (Na V ) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa , is the first reported plant-derived Na V channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at Na V channels, and that co-expression of TMEM233 modulates the gating properties of Na V 1.7. These findings identify TMEM233 as a previously unknown Na V 1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on Na V channel gating, and provide important insights into the function of Na V channels in sensory neurons.
Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels.
Publisher: Springer Science and Business Media LLC
Date: 28-09-2016
DOI: 10.1038/SREP34333
Abstract: Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.
Cyclization of Peptides by using Selenolanthionine Bridges
Publisher: Wiley
Date: 18-09-2012
Two proteins for the price of one: Structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1
Publisher: Elsevier BV
Date: 07-2017
Structural basis for the binding of the cancer targeting scorpion toxin, ClTx, to the vascular endothelia growth factor receptor neuropilin-1
Publisher: Elsevier BV
Date: 2021
Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics
Publisher: Informa UK Limited
Date: 22-12-2018
Bacillus anthracis Protective Antigen Shows High Specificity for a UV Induced Mouse Model of Cutaneous Squamous Cell Carcinoma
Publisher: Frontiers Media SA
Date: 12-02-2019
Site-specific pK a determination of selenocysteine residues in selenovasopressin by using 77Se NMR spectroscopy
Publisher: Wiley
Date: 14-10-2011
Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases
Publisher: Proceedings of the National Academy of Sciences
Date: 21-04-2011
Abstract: Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.
A tarantula-venom peptide antagonizes the TRPA1 nociceptor Ion channel by binding to the S1-S4 gating domain
Publisher: Elsevier BV
Date: 03-2014
Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa
Publisher: Elsevier BV
Date: 10-2019
DOI: 10.1016/J.TOXICON.2019.07.002
Abstract: Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D
Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain
Publisher: Elsevier BV
Date: 03-2012
DOI: 10.1016/J.BIOCHI.2011.09.012
Abstract: The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690-704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform s ling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation h ered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution - an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins.
Solution structure of the RNA-binding cold shock domain of the Chlamydomonas reinhardtii NAB1 protein and insights into RNA recognition
Publisher: Portland Press Ltd.
Date: 19-06-2015
DOI: 10.1042/BJ20150217
Abstract: Light-harvesting complex (LHC) proteins are among the most abundant proteins on Earth and play critical roles in photosynthesis, both in light capture and in photoprotective mechanisms. The Chlamydomonas reinhardtii nucleic acid-binding protein 1 (NAB1) is a negative regulator of LHC protein translation. Its N-terminal cold-shock domain (CSD) binds to a 13-nt element [CSD consensus sequence (CSDCS)] found in the mRNA of specific LHC proteins associated with Photosystem II (PSII), an interaction which regulates LHC expression and, consequently, PSII-associated antenna size, structure and function. In the present study, we elucidated the solution structure of the NAB1 CSD as determined by heteronuclear NMR. The CSD adopts a characteristic five-stranded anti parallel β-barrel fold. Upon addition of CSDCS RNA, a large number of NMR chemical shift perturbations were observed, corresponding primarily to surface-exposed residues within the highly conserved β2- and β3-strands in the canonical RNA-binding region, but also to residues on β-strand 5 extending the positive surface patch and the overall RNA-binding site. Additional chemical shift perturbations that accompanied RNA binding involved buried residues, suggesting that transcript recognition is accompanied by conformational change. Our results indicate that NAB1 associates with RNA transcripts through a mechanism involving its CSD that is conserved with mechanisms of sequence-specific nucleic acid recognition employed by ancestrally related bacterial cold-shock proteins (CSPs).
NMR methods for determining disulfide-bond connectivities
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.TOXICON.2010.06.018
Abstract: Animal toxins are the major class of secreted disulfide-rich proteins, with approximately 70% containing two or more disulfide bonds. Incorrect pairing of these disulfide bonds typically leads to a non-native three-dimensional fold accompanied by a loss of protein function. Determination of the native disulfide-bond framework is therefore a key component in the structural characterization of toxins. In this article, we review NMR approaches for elucidation of disulfide-bond connectivities. A major advantage of these NMR approaches is that they are non-invasive, leaving the s le intact at the end of the analysis for use in other studies.
Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target Na(V)1.7
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 10-2015
Abstract: 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.
Macromolecular NMR spectroscopy for the non-spectroscopist: Beyond macromolecular solution structure determination
Publisher: Wiley
Date: 28-01-2011
DOI: 10.1111/J.1742-4658.2011.08005.X
Abstract: A strength of NMR spectroscopy is its ability to monitor, on an atomic level, molecular changes and interactions. In this review, which is intended for non-spectroscopist, we describe major uses of NMR in protein science beyond solution structure determination. After first touching on how NMR can be used to quickly determine whether a mutation induces structural perturbations in a protein, we describe the unparalleled ability of NMR to monitor binding interactions over a wide range of affinities, molecular masses and solution conditions. We discuss the use of NMR to measure the dynamics of proteins at the atomic level and over a wide range of timescales. Finally, we outline new and expanding areas such as macromolecular structure determination in multicomponent systems, as well as in the solid state and in vivo.
Backbone and side chain NMR assignments of Geobacillus stearothermophilus ZapA allow identification of residues that mediate the interaction of ZapA with FtsZ
Publisher: Springer Science and Business Media LLC
Date: 13-05-2015
DOI: 10.1007/S12104-015-9615-1
Abstract: Bacterial ision 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 ision 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.
Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6
Publisher: Wiley
Date: 28-04-2014
DOI: 10.1111/FEBS.12800
Abstract: Human interleukin (IL)-6 plays a pivotal role in the immune response, hematopoiesis, the acute-phase response, and inflammation. IL-6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL-6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α-receptor [IL-6 α-receptor (IL-6R)] via site I. Here, we present the backbone dynamics of apo-IL-6 as determined by analysis of NMR relaxation data with the extended model-free formalism of Lipari and Szabo. To alleviate significant resonance overlap in the HSQC-type spectra, cell-free protein synthesis was used to selectively (15) N-label residues, thereby ensuring a complete set of residue-specific dynamics. The calculated order parameters [square of the generalized model-free order parameter (S(2))] showed significant conformational heterogeneity among clusters of residues in IL-6. In particular, the N-terminal region of the long AB-loop, which corresponds spatially to one of the gp130 receptor binding epitopes (i.e. site III), experiences substantial fluctuations along the conformation of the main chain (S(2) = 0.3-0.8) that are not observed at the other two epitopes or in other cytokines. Thus, we postulate that dynamic properties of the AB-loop are responsible for inhibiting the interaction of IL-6 with gp130 in the absence of the IL-6R, and that binding of IL-6R at site I shifts the dynamic equilibrium to favor interaction with gp130 at site III. In addition, molecular dynamics simulations corroborated the NMR-derived dynamics, and showed that the BC-loop adopts different substates that possibly play a role in facilitating receptor assembly.
Elucidating the Lipid Binding Properties of Membrane-Active Peptides Using Cyclised Nanodiscs
Publisher: Frontiers Media SA
Date: 16-04-2019
Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR
Publisher: Copernicus GmbH
Date: 29-11-2022
DOI: 10.5194/MR-2022-22
Abstract: Abstract. Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (atb.uq.edu.au) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of sidechains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.
Toxin structures as evolutionary tools: Using conserved 3D folds to study the evolution of rapidly evolving peptides
Publisher: Wiley
Date: 11-05-2016
Abstract: Three-dimensional (3D) structures have been used to explore the evolution of proteins for decades, yet they have rarely been utilized to study the molecular evolution of peptides. Here, we highlight areas in which 3D structures can be particularly useful for studying the molecular evolution of peptide toxins. Although we focus our discussion on animal toxins, including one of the most widespread disulfide-rich peptide folds known, the inhibitor cystine knot, our conclusions should be widely applicable to studies of the evolution of disulfide-constrained peptides. We show that conserved 3D folds can be used to identify evolutionary links and test hypotheses regarding the evolutionary origin of peptides with extremely low sequence identity construct accurate multiple sequence alignments and better understand the evolutionary forces that drive the molecular evolution of peptides. Also watch the video abstract.
Weaponization of a Hormone: Convergent Recruitment of Hyperglycemic Hormone into the Venom of Arthropod Predators
Publisher: Elsevier BV
Date: 07-2015
DOI: 10.1016/J.STR.2015.05.003
Abstract: Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through ergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution.
The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation
Publisher: Public Library of Science (PLoS)
Date: 13-01-2011
A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.PEP.2019.04.004
Abstract: We have developed a new ligation independent cloning (LIC) vector - pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHis-tag and the catalytic core of SrtA from Staphylococcus aureus (SrtAΔ59). Thus, after cloning and protein expression in Escherichia coli, the resultant fusion protein comprises an N-terminal 6xHis-tag, SrtAΔ59, an L-P-E-T-G linker and the protein of interest at the C-terminus. The fusion protein can be captured onto immobilized Ni-NTA resin and any unwanted proteolysis activity of SrtA is suppressed during the purification by optimisation of solution conditions. Upon addition of Ca
Structural insights into the mechanism of inhibition of AHAS by herbicides
Publisher: Proceedings of the National Academy of Sciences
Date: 13-02-2018
Abstract: Acetohydroxyacid synthase (AHAS), the first enzyme in the branched amino acid biosynthesis pathway, is present only in plants and microorganisms, and it is the target of commercial herbicides. Penoxsulam (PS), which is a highly effective broad-spectrum AHAS-inhibiting herbicide, is used extensively to control weed growth in rice crops. However, the molecular basis for its inhibition of AHAS is poorly understood. This is despite the availability of structural data for all other classes of AHAS-inhibiting herbicides. Here, crystallographic data for Saccharomyces cerevisiae AHAS (2.3 Å) and Arabidopsis thaliana AHAS (2.5 Å) in complex with PS reveal the extraordinary molecular mechanisms that underpin its inhibitory activity. The structures show that inhibition of AHAS by PS triggers expulsion of two molecules of oxygen bound in the active site, releasing them as substrates for an oxygenase side reaction of the enzyme. The structures also show that PS either stabilizes the thiamin diphosphate (ThDP)-peracetate adduct, a product of this oxygenase reaction, or traps within the active site an intact molecule of peracetate in the presence of a degraded form of ThDP: thiamine aminoethenethiol diphosphate. Kinetic analysis shows that PS inhibits AHAS by a combination of events involving FAD oxidation and chemical alteration of ThDP. With the emergence of increasing levels of resistance toward front-line herbicides and the need to optimize the use of arable land, these data suggest strategies for next generation herbicide design.
ScrepYard: an online resource for disulfide-stabilised tandem repeat peptides
Publisher: Cold Spring Harbor Laboratory
Date: 20-01-2022
DOI: 10.1101/2022.01.17.476686
Abstract: Receptor avidity through multivalency is a highly sought-after property of ligands. While readily available in nature in the form of bivalent antibodies, this property remains challenging to engineer in synthetic molecules. The discovery of several bivalent venom peptides containing two homologous and independently folded domains (in a tandem repeat arrangement) has provided a unique opportunity to better understand the underpinning design of multivalency in multimeric biomolecules, as well as how naturally occurring multivalent ligands can be identified. In previous work we classified these molecules as a larger class termed secreted cysteine-rich repeat-proteins (SCREPs). Here, we present an online resource ScrepYard, designed to assist researchers in identification of SCREP sequences of interest and to aid in characterizing this emerging class of biomolecules. Analysis of sequences within the ScrepYard reveals that two-domain tandem repeats constitute the most abundant SCREP domain architecture, while the interdomain “linker” regions connecting the ordered domains are found to be abundant in amino acids with short or polar sidechains and contain an unusually high abundance of proline residues. Finally, we demonstrate the utility of ScrepYard as a virtual screening tool for discovery of putatively multivalent peptides, by using it as a resource to identify a previously uncharacterised serine protease inhibitor and confirm its predicated activity using an enzyme assay.
The tarantula toxin β/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity
Publisher: Springer Science and Business Media LLC
Date: 20-04-2017
DOI: 10.1038/S41598-017-01129-0
Abstract: Voltage-gated sodium (Na V ) 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 Na V channels inhibiting peak current of hNa V 1.1, rNa V 1.2, hNa V 1.6, and hNa V 1.7 while concurrently inhibiting fast inactivation of hNa V 1.1 and rNa V 1.3. The DII and DIV S3-S4 loops of Na V channel voltage sensors are important for the interaction of Pre1a with Na V 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 Na V 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 Na V 1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of Na V channels may be necessary for functional modulation, and that targeting the erse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.
Direct Visualization of Disulfide Bonds through Diselenide Proxies Using 77Se NMR Spectroscopy
Publisher: Wiley
Date: 17-11-2009
α-Conotoxin Dendrimers Have Enhanced Potency and Selectivity for Homomeric Nicotinic Acetylcholine Receptors
Publisher: American Chemical Society (ACS)
Date: 03-03-2015
DOI: 10.1021/JACS.5B00244
Abstract: Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.
A non-uniform sampling approach enables studies of dilute and unstable proteins
Publisher: Springer Science and Business Media LLC
Date: 10-02-2017
DOI: 10.1007/S10858-017-0091-Z
Abstract: NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on s le preparation and the high level of control of s le conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated s les or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform s ling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein s le that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform s ling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.
Understanding the molecular basis of toxin promiscuity: The analgesic sea anemone peptide APETx2 interacts with acid-sensing ion channel 3 and hERG channels via overlapping pharmacophores
Publisher: American Chemical Society (ACS)
Date: 04-11-2014
DOI: 10.1021/JM501400P
Abstract: The sea anemone peptide APETx2 is a potent and selective blocker of acid-sensing ion channel 3 (ASIC3). APETx2 is analgesic in a variety of rodent pain models, but the lack of knowledge of its pharmacophore and binding site on ASIC3 has impeded development of improved analogues. Here we present a detailed structure-activity relationship study of APETx2. Determination of a high-resolution structure of APETx2 combined with scanning mutagenesis revealed a cluster of aromatic and basic residues that mediate its interaction with ASIC3. We show that APETx2 also inhibits the off-target hERG channel by reducing the maximal current litude and shifting the voltage dependence of activation to more positive potentials. Electrophysiological screening of selected APETx2 mutants revealed partial overlap between the surfaces on APETx2 that mediate its interaction with ASIC3 and hERG. Characterization of the molecular basis of these interactions is an important first step toward the rational design of more selective APETx2 analogues.
The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop
Publisher: Wiley
Date: 23-01-2015
DOI: 10.1111/FEBS.13189
Abstract: Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.
Venomics: A new paradigm for natural products-based drug discovery
Publisher: Springer Science and Business Media LLC
Date: 24-02-2011
DOI: 10.1007/S00726-010-0516-4
Abstract: The remarkable potency and pharmacological ersity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical ersity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular ersity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.
A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a
Publisher: Springer Science and Business Media LLC
Date: 11-07-2014
DOI: 10.1038/NCOMMS5350
Functional expression in Escherichia coli of the disulfide-richsea anemone peptide APETx2, a potent blocker of acid-sensing ion channel 3
Publisher: MDPI AG
Date: 23-07-2012
DOI: 10.3390/MD10071605
The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant
Publisher: Public Library of Science (PLoS)
Date: 16-03-2017
Inhibition of the norepinephrine transporter by χ-conotoxin dendrimers
Publisher: Wiley
Date: 22-02-2016
DOI: 10.1002/PSC.2857
Abstract: Peptide dendrimers are a novel class of macromolecules of emerging interest with the potential of delayed renal clearance due to their molecular size and enhanced activity due to the multivalency effect. In this work, an active analogue of the disulfide-rich χ-conotoxin χ-MrIA (χ-MrIA), a norepinephrine reuptake (norepinephrine transporter) inhibitor, was grafted onto a polylysine dendron. Dendron decoration was achieved by employing copper-catalyzed alkyne-azide cycloaddition with azido-PEG chain-modified χ-MrIA analogues, leading to homogenous 4-mer and 8-mer χ-MrIA dendrimers with molecular weights ranging from 8 to 22 kDa. These dendrimers were investigated for their impact on peptide secondary structure, in vitro functional activity, and potential anti-allodynia in vivo. NMR studies showed that the χ-MrIA tertiary structure was maintained in the χ-MrIA dendrimers. In a functional norepinephrine transporter reuptake assay, χ-MrIA dendrimers showed slightly increased potency relative to the azido-PEGylated χ-MrIA analogues with similar potency to the parent peptide. In contrast to χ-MrIA, no anti-allodynic action was observed when the χ-MrIA dendrimers were administered intrathecally in a rat model of neuropathic pain, suggesting that the larger dendrimer structures are unable to diffuse through the spinal column tissue and reach the norepinephrine transporter. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria
Publisher: Springer Science and Business Media LLC
Date: 23-06-2020
DOI: 10.1038/S41467-020-16950-X
Abstract: Peptide antibiotics are an abundant and synthetically tractable source of molecular ersity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an hipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina . The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC , a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3–4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the ‘no observable adverse effect level’ (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.
The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins
Publisher: Proceedings of the National Academy of Sciences
Date: 17-10-2016
Abstract: Plants and animals use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to defend themselves against invading microbes. In this study, we report the solution structure of the N-terminal coiled-coil (CC) domain from the wheat stem rust resistance protein Sr33. Remarkably, this structure differs substantially from the published crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein MLA10. Using a structural, biophysical, and functional approach, we compare the Sr33 CC domain with other structurally defined NLR CC domains. Collectively, this work redefines our current understanding of the structure and function of plant NLR CC domains, which has significant implications for future studies into this important class of defense receptors.
Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type CaV channels
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1016/J.BCP.2014.02.008
Abstract: Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels.
A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.
Publisher: Springer Science and Business Media LLC
Date: 23-02-2023
DOI: 10.1038/S41467-023-36579-W
Abstract: Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis , a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The in idual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca 2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.
Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling
Publisher: Proceedings of the National Academy of Sciences
Date: 24-07-2017
Abstract: Toll-like receptor (TLR) signaling pathways are targeted to limit inflammation in immune cells. TLRs use adaptor proteins to drive inflammatory signaling platforms for effective microbial clearance. Here we show that MyD88 adaptor-like (MAL), an adaptor protein in TLR signaling, undergoes glutathionylation in response to LPS, driving macrophage responses to proinflammatory stimuli. We also determined the solution structure of MAL in the reduced form without disulfides, revealing a typical BB loop observed in adaptor proteins, in contrast to previously reported crystal structures. This alternate solution structure reveals the inherent flexibility of MAL, supporting the hypothesis that glutathionylation may reposition the MAL BB loop for MyD88 interaction to drive inflammation. This discovery could lead to novel approaches to target MAL glutathionylation in dysregulated TLR signaling, limiting inflammation.
Isolation of an Orally Active Insecticidal Toxin from the Venom of an Australian Tarantula
Publisher: Public Library of Science (PLoS)
Date: 11-09-2013
Effective Protocol for Database Similarity Searching of Heteronuclear Single Quantum Coherence Spectra
Publisher: American Chemical Society (ACS)
Date: 26-10-2009
DOI: 10.1021/AC901616T
Abstract: The nuclear magnetic resonance (NMR) chemical shift exquisitely describes the chemical environment of the atoms in a molecule. Here we describe methods that utilize this information as an experimental probe to match 2D NMR heteronuclear single quantum coherence (HSQC) spectra of pure, unknown compounds to a database of known compounds. We implemented and compared two different approaches for similarity searching of HSQC spectra. According to our findings, our new discrete self-adaptive differential evolution method performs better than the previously published shifted grid, multiple resolution approach. The new method is provided in detail and comparisons have been performed for a set of HSQC spectra. The similarity comparison involves a peak-to-peak matching of different spectra, followed by a selection criterion and ranking to establish a level of match.
Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation
Publisher: Springer Science and Business Media LLC
Date: 04-03-2023
DOI: 10.1038/S42004-023-00841-5
Abstract: Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery—either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Recent advances in enzyme-catalysed macrocyclisation include discovery of new enzymes or design of new engineered enzymes. Here, we describe the engineering of a self-cyclising “ autocyclase ” protein, capable of performing a controllable unimolecular reaction for generation of cyclic biomolecules in high yield. We characterise the self-cyclisation reaction mechanism, and demonstrate how the unimolecular reaction path provides alternative avenues for addressing existing challenges in enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple, alternative way to access a vast ersity of macrocyclic biomolecules.
Evaluation of Chemical Strategies for Improving the Stability and Oral Toxicity of Insecticidal Peptides
Publisher: MDPI AG
Date: 28-08-2018
DOI: 10.3390/BIOMEDICINES6030090
Abstract: Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been h ered 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 ω-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.
RNA polymerase-induced remodelling of NusA produces a pause enhancement complex
Publisher: Oxford University Press (OUP)
Date: 17-02-2015
DOI: 10.1093/NAR/GKV108
Efficient biosynthesis of heterodimeric C3-aryl pyrroloindoline alkaloids
Publisher: Springer Science and Business Media LLC
Date: 24-10-2018
DOI: 10.1038/S41467-018-06528-Z
Abstract: Many natural products contain the hexahydropyrrolo[2, 3- b ]indole (HPI) framework. HPI containing chemicals exhibit various biological activities and distinguishable structural arrangement. This structural complexity renders chemical synthesis very challenging. Here, through investigating the biosynthesis of a naturally occurring C 3 -aryl HPI, naseseazine C (NAS-C), we identify a P450 enzyme (NascB) and reveal that NascB catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon–carbon bonds with both regio- and stereo-specificity. Surprisingly, the limited freedom is allowed in specificity to generate four types of C 3 -aryl HPI scaffolds, and two of them were not previously observed. By incorporating NascB into an engineered strain of E. coli , we develop a whole-cell biocatalysis system for efficient production of NAS-C and 30 NAS analogs. Interestingly, we find that some of these analogs exhibit potent neuroprotective properties. Thus, our biocatalytic methodology offers an efficient and simple route to generate difficult HPI framework containing chemicals.
Synthesis of Multivalent [Lys(8)]-Oxytocin Dendrimers that Inhibit Visceral Nociceptive Responses
Publisher: CSIRO Publishing
Date: 2017
DOI: 10.1071/CH16407
Abstract: Peptide dendrimers are a novel class of precisely defined macromolecules of emerging interest. Here, we describe the synthesis, structure, binding affinity, receptor selectivity, functional activity, and antinociceptive properties of oxytocin-related dendrimers containing up to 16 copies of [Lys8]-oxytocin or LVT. These were generated using a copper(i)-catalyzed azide–alkyne cycloaddition (CuAAc) reaction with azido-pegylated LVT peptides on an alkyne–polylysine scaffold. 2D NMR analysis demonstrated that each attached LVT ligand was freely rotating and maintained identical 3D structures in each dendrimeric macromolecule. The binding affinity Ki at the oxytocin receptor increased approximately 17-, 12-, 3-, and 1.5-fold respectively for the 2-, 4-, 8-, and 16-mer dendrimeric LVT conjugates, compared with monomer azido-pegylated LVT (Ki = 9.5 nM), consistent with a multivalency effect. A similar trend in affinity was also observed at the related human V1a, V1b, and V2 receptors, with no significant selectivity change observed across this family of receptors. All LVT dendrimers were functionally active in vitro on human oxytocin receptors and inhibited colonic nociceptors potently in a mouse model of chronic abdominal pain.
Do Vicinal Disulfide Bridges Mediate Functionally Important Redox Transformations in Proteins?
Publisher: Mary Ann Liebert Inc
Date: 12-2013
Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene.
Publisher: Proceedings of the National Academy of Sciences
Date: 12-05-2020
Abstract: 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 erse 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 % 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.
Chemical synthesis, 3D structure, and ASIC binding site of the toxin mambalgin-2
Publisher: Wiley
Date: 09-12-2014
Abstract: Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide.
Video with Impact: Access to the World’s Magnetic-Resonance Experts for the Scientific-Education Community
Publisher: American Chemical Society (ACS)
Date: 03-12-2018
Macromolecular NMR spectroscopy for the non-spectroscopist
Publisher: Wiley
Date: 28-01-2011
DOI: 10.1111/J.1742-4658.2011.08004.X
Abstract: NMR spectroscopy is a powerful tool for studying the structure, function and dynamics of biological macromolecules. However, non-spectroscopists often find NMR theory daunting and data interpretation nontrivial. As the first of two back-to-back reviews on NMR spectroscopy aimed at non-spectroscopists, the present review first provides an introduction to the basics of macromolecular NMR spectroscopy, including a discussion of typical s le requirements and what information can be obtained from simple NMR experiments. We then review the use of NMR spectroscopy for determining the 3D structures of macromolecules and examine how to judge the quality of NMR-derived structures.
Modulation of Ion Channels by Cysteine-Rich Peptides
Publisher: Elsevier
Date: 2017
DOI: 10.1016/BS.APHA.2017.03.001
Abstract: Venom peptides are natural ligands of ion channels and have been used extensively in pharmacological characterization of various ion channels and receptors. In this chapter, we survey all known venom peptide ion-channel modulators. Our survey reveals that the majority of venom peptides characterized to date target voltage-gated sodium or potassium channels. We further find that the majority of these peptides are found in scorpion and spider venoms. We discuss the influence of the pharmacological tools available in biasing discovery and the classical "toxin-to-sequence" approach to venom peptide biodiscovery. The impact of high-throughput sequencing on the existing discovery framework is likely to be significant and we propose here an alternative "sequence-to-toxin" approach to peptide screening, relying more on recently developed high-throughput methods. Methods for production and characterization of disulfide rich toxins in a high-throughput setting are then described, focusing on bacterial protein expression and solution state structural characterization by NMR spectroscopy. Finally, the role of X-ray crystallography and cryo-EM are highlighted by discussing the currently known channel-peptide complexes.
A non-uniformly sampled 4D HCC(CO)NH-TOCSY experiment processed using maximum entropy for rapid protein sidechain assignment
Publisher: Elsevier BV
Date: 05-2010
Development of a μO-conotoxin analogue with improved lipid membrane interactions and potency for the analgesic sodium channel NaV 1.8
Publisher: Elsevier BV
Date: 05-2016
Chemical synthesis and structure of the prokineticin Bv8
Publisher: Wiley
Date: 30-08-2010
Abstract: Bv8, a 77-residue protein isolated from frogs, is the prototypic member of the prokineticin family of cytokines. Prokineticins (PKs) have only recently been identified in vertebrates (including humans), and they are believed to be involved in a number of key physiological processes, such as angiogenesis, neurogenesis, nociception, and tissue development. We used a combination of Boc solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited full activity in a human neuroblastoma cell line and rat dorsal root ganglion (DRG) neurons. The 3D structure of the synthetic protein was determined by using NMR spectroscopy and it was found to be homologous with that of mamba intestinal toxin 1, which is the only other known prokineticin structure. Analysis of a truncated mutant lacking five residues at the N terminus that are critical for receptor binding and activation showed no perturbation to the core protein structure. Together with the functional data, this suggests that receptor binding is likely to be a highly cooperative process possibly involving major allosterically driven structural rearrangements. The facile and efficient synthesis presented here will enable preparation of unique chemical analogues of prokineticins, which should be powerful tools for modulating the structure and function of prokineticins and their receptors, and studying the many physiological processes that have been linked to them.
NaV1.7 as a pain target – From gene to pharmacology
Publisher: Elsevier BV
Date: 04-2017
Selective Na V 1.1 activation rescues Dravet syndrome mice from seizures and premature death
Publisher: Proceedings of the National Academy of Sciences
Date: 03-08-2018
Abstract: Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit Na V 1.1. Cell-based assays showed Hm1a was selective for hNa V 1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with Na V 1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders.
Multi-species transcriptomics reveals evolutionary diversity in the mechanisms regulating shrimp tail muscle excitation-contraction coupling
Publisher: Elsevier BV
Date: 08-2020
Related Organisations
University Of Liverpool
Location: United Kingdom of Great Britain and Northern Ireland
Related Funding Activities
ARC Future Fellowships - Grant ID: FT110100925
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End Date: 05-2016
Amount: $660,028.00
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End Date: 01-2022
Amount: $414,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP220103028
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End Date: 01-2025
Amount: $431,873.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP140101098
Start Date: 2014
End Date: 01-2017
Amount: $150,000.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE230100048
Start Date: 03-2023
End Date: 03-2024
Amount: $570,702.00
Funder: Australian Research Council
View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE160100218
Start Date: 2016
End Date: 12-2016
Amount: $840,000.00
Funder: Australian Research Council
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