ABSTRACT
Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.
Subject(s)
Toxins, Biological , Animals , Peptides , Electrophysiological PhenomenaABSTRACT
Numerous spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by reversed-phase high-performance liquid chromatography (RP-HPLC). This conformational exchange is especially peculiar for peptides containing an inhibitor cystine knot motif, which confers excellent structural stability under conditions that are not conducive to disulfide shuffling. This phenomenon is often attributed to proline cis/trans isomerization but has also been observed in peptides that do not contain a proline residue. Pn3a is one such peptide forming two chromatographically distinguishable peaks that readily interconvert following the purification of either conformer. The nature of this exchange was previously uncharacterized due to the fast rate of conversion in solution, making isolation of the conformers impossible. In the present study, an N-terminal modification of Pn3a enabled the isolation of the individual conformers, allowing activity assays to be conducted on the individual conformers using electrophysiology. The conformers were analyzed separately by nuclear magnetic resonance spectroscopy (NMR) to study their structural differences. RP-HPLC and NMR were used to study the mechanism of exchange. The later-eluting conformer was the active conformer with a rigid structure that corresponds to the published structure of Pn3a, while NMR analysis revealed the earlier-eluting conformer to be inactive and disordered. The exchange was found to be pH-dependent, arising in acidic solutions, possibly due to reversible disruption and formation of intramolecular salt bridges. This study reveals the nature of non-proline conformational exchange observed in Pn3a and possibly other disulfide-rich peptides, highlighting that the structure and activity of some disulfide-stabilized peptides can be dramatically susceptible to disruption.
ABSTRACT
Voltage-gated sodium (NaV) 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 NaV 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 NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.
Subject(s)
Toxins, Biological , Urtica dioica , Australia , Pain , Peptides , NAV1.7 Voltage-Gated Sodium Channel/metabolismABSTRACT
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 diversity of macrocyclic biomolecules.
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 individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ 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.
Subject(s)
Ryanodine Receptor Calcium Release Channel , Scorpion Venoms , Ryanodine Receptor Calcium Release Channel/genetics , Ryanodine Receptor Calcium Release Channel/metabolism , Calcium/metabolism , Ryanodine/pharmacology , Amino Acid Sequence , Peptides/chemistry , Scorpion Venoms/pharmacology , Scorpion Venoms/chemistryABSTRACT
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 functional 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 uncharacterized serine protease inhibitor and confirm its predicted activity using an enzyme assay.
Subject(s)
Disulfides , Peptides , Amino Acid Sequence , Peptides/chemistry , Tandem Repeat Sequences , Amino AcidsABSTRACT
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 Ca2+ and triglycine (Gly3), the immobilized fusion protein undergoes on-column SrtA-mediated cleavage at the T-G bond of LPETG linker to selectively release 90% of the protein of interest within 3â¯h when incubated at room temperature. This new pSrtA9 vector, thus, offers an efficient method for LIC of genes and a one-step purification procedure to obtain a tag-free recombinant protein, and is therefore suitable for the high-throughput proteins production.
Subject(s)
Aminoacyltransferases/genetics , Bacterial Proteins/genetics , Cloning, Molecular/methods , Cysteine Endopeptidases/genetics , Genetic Vectors/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Amino Acid Motifs , Aminoacyltransferases/chemistry , Aminoacyltransferases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/metabolism , Recombinant Fusion Proteins/metabolism , Staphylococcus aureus/geneticsABSTRACT
Squamous cell carcinoma (SCC) accounts for the majority of non-melanoma skin cancer related deaths, particularly in immunosuppressed persons. Identification of biomarkers that could be used to identify or treat SCC would be of significant benefit. The anthrax toxin receptors, Tumor Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2), are endothelial receptors involved in extracellular matrix homeostasis and angiogenesis that are selectively upregulated on numerous tumors. One method of targeting these receptors is Protective Antigen (PA), a protein produced by B. anthracis that mediates binding and translocation of anthrax toxins into cells. PA targeted toxins have been demonstrated to selectively inhibit tumor growth and angiogenesis, but tumor selectivity of PA is currently unknown. In this work fluorescently labeled PA was shown to maintain receptor dependent binding and internalization in vitro. Utilizing a human papillomavirus transgenic mouse model that develops cutaneous SCC in response to ultraviolet irradiation we identified tumor uptake of PA in vivo. The intravenously administered PA resulted in tumor specific localization, with exclusive tumor detection 24 h post injection. Ex vivo analysis identified significantly higher fluorescence in the tumor compared to adjacent healthy tissue and major clearance organs, demonstrating low non-specific uptake and rapid clearance. While both TEM8 and CMG2 were observed to be overexpressed in SCC tumor sections compared to control skin, the intravenously administered PA was primarily co-localized with TEM8. These results suggest that PA could be systemically administered for rapid identification of cutaneous SCC, with potential for further therapeutic development.