Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.
Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.membrane trafficking | protein-peptide interaction | protein-protein interaction | Sec/Munc protein S ec/Munc (SM) and soluble NSF attachment protein receptor (SNARE) proteins play fundamental roles in regulating membrane traffic (1-3). The cognate interacting partners comprising the SM protein Munc18-1 and the SNARE protein Syntaxin1 (Sx1) are of special importance to human physiology because they regulate synaptic vesicle-mediated neurotransmitter release (4). Two alternate binding modes have been described for this pair of proteins. One mode involves Munc18-1 interacting with "closed" Sx1, in which the SNARE H3 helical motif is sequestered by the three Habc helices of Sx1 to form a four-helix bundle (5, 6) ( Fig. 1). This closed binding mode is consistent with a negative regulatory role for Munc18-1 because the SNARE H3 helix in closed Sx1 is unable to interact with SNARE partners to form the complexes that drive vesicle fusion (7) (Fig. 1A). However, the closed binding mode of Syntaxin is not universal and may be a specialization of regulated exocytosis (8). A second binding mode, which likely underpins a general function of SM proteins, occurs when Sx1 is in an "open" conformation (i.e., when the H3 helix is separated from the Habc helices) in the SNARE ternary complex (9, 10) (Fig. 1A). This binding mode is dependent on the very N-terminal 10 residues of Sx1, the N peptide. This second mode is consistent with a positive regulatory role for Munc18-1, because SNARE ternary complex formation is required for vesicle fusion. The N-peptide interaction has been characterized structurally for the highly homologous protein pair of Munc18-3 and Syntaxin4 (Sx4) (11), which regulate trafficking of the insulin-stimulated glucose transporter GLUT4 in muscle and fat cells (12).Munc18 proteins contribute to s...
Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2–6 disulfide bonds.
Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is -TRTXPc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. -TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the -TRTX-Pc1a-ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of -TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a -hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of -TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful -TRTX-Pc1a mimetics.
Supplementary data are available at Bioinformatics online.
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