Richard B. Jacobsen

α-Conotoxin AuIB Selectively Blocks α3β4 Nicotinic Acetylcholine Receptors and Nicotine-Evoked Norepinephrine Release

Neuronal nicotinic acetylcholine receptors (nAChRs) with putative α3β4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of α3β4-selective ligands. In this report, we describe the purification and characterization of an α3β4 nAChR antagonist, α-conotoxin AuIB, from the venom of the “court cone,”Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. α-Conotoxin AuIB blocks α3β4 nAChRs expressed in Xenopus oocytes with an IC50 of 0.75 μm, a kon of 1.4 × 106 min-1m−1, a koffof 0.48 min-1, and aKd of 0.5 μm. Furthermore, α-conotoxin AuIB blocks the α3β4 receptor with >100-fold higher potency than other receptor subunit combinations, including α2β2, α2β4, α3β2, α4β2, α4β4, and α1β1γδ. Thus, AuIB is a novel, selective probe for α3β4 nAChRs. AuIB (1–5 μm) blocks 20–35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the α3β2-specific α-conotoxin MII (100 nm) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, α3β4-containing nAChRs mediate norepinephrine release, whereas α3β2-containing receptors mediate dopamine release.

μ-Conotoxin PIIIA, a New Peptide for Discriminating among Tetrodotoxin-Sensitive Na Channel Subtypes

We report the characterization of a new sodium channel blocker, μ-conotoxin PIIIA (μ-PIIIA). The peptide has been synthesized chemically and its disulfide bridging pattern determined. The structure of the new peptide is: where Z = pyroglutamate andO = 4-trans-hydroxyproline. We demonstrate that Arginine-14 (Arg14) is a key residue; substitution by alanine significantly decreases affinity and results in a toxin unable to block channel conductance completely. Thus, like all toxins that block at Site I, μ-PIIIA has a critical guanidinium group. This peptide is of exceptional interest because, unlike the previously characterized μ-conotoxin GIIIA (μ-GIIIA), it irreversibly blocks amphibian muscle Na channels, providing a useful tool for synaptic electrophysiology. Furthermore, the discovery of μ-PIIIA permits the resolution of tetrodotoxin-sensitive sodium channels into three categories: (1) sensitive to μ-PIIIA and μ-conotoxin GIIIA, (2) sensitive to μ-PIIIA but not to μ-GIIIA, and (3) resistant to μ-PIIIA and μ-GIIIA (examples in each category are skeletal muscle, rat brain Type II, and many mammalian CNS subtypes, respectively). Thus, μ-conotoxin PIIIA provides a key for further discriminating pharmacologically among different sodium channel subtypes.

The T-superfamily of conotoxins.

We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conusspecies were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conusspecies. These peptides are likely to be functionally diverse; although the peptides are small (11–17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, γCCγDGW+CCT§AAO; and au5a, FCCPFIRYCCW (where γ = γ-carboxyglutamate, W+ = bromotryptophan, O = hydroxyproline, T§ = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional γ-carboxylation recognition signal in the −1 to −20 propeptide region, consistent with the presence of γ-carboxyglutamate residues in this peptide.

Single Amino Acid Substitutions in κ-Conotoxin PVIIA Disrupt Interaction with the Shaker K+ Channel

κ-Conotoxin PVIIA (κ-PVIIA), a 27-amino acid peptide with three disulfide cross-links, isolated from the venom ofConus purpurascens, is the first conopeptide shown to inhibit the Shaker K+ channel (Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148–151). Recently, two groups independently determined the solution structure for κ-PVIIA using NMR; although the structures reported were similar, two mutually exclusive models for the interaction of the peptide with theShaker channel were proposed. We carried out a structure/function analysis of κ-PVIIA, with alanine substitutions for all amino acids postulated to be key residues by both groups. Our data are consistent with the critical dyad model developed by Ménez and co-workers (Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, B., Roumestand, C., Vita, C., de Medeiros, C., Rowan, E. G., Harvey, A. L., and Ménez, A. (1997)J. Biol. Chem. 272, 4802–4809) for polypeptide antagonists of K+ channels. In the case of κ-PVIIA, Lys7 and Phe9 are essential for activity as predicted by Savarin et al. (Savarin, P., Guenneugues, M., Gilquin, B., Lamthanh, H., Gasparini, S., Zinn-Justin, S., and Ménez, A. (1998) Biochemistry 37, 5407–5416); these workers also correctly predicted an important role for Lys25. Thus, although κ-conotoxin PVIIA has no obvious sequence homology to polypeptide toxins from other venomous animals that interact with voltage-gated K+ channels, there may be convergent functional features in diverse K+ channel polypeptide antagonists.

Escherichia coli Signal Peptide Peptidase A Is a Serine-Lysine Protease with a Lysine Recruited to the Nonconserved Amino-Terminal Domain in the S49 Protease Family†

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.

The interfacial bioscience grand challenge.

This report is broken down into the following 3 sections: (1) Chemical Cross-linking and Mass Spectrometry Applied to Determination of Protein Structure and Dynamics; (2) Computational Modeling of Membrane Protein Structure and Dynamics; and (3) Studies of Toxin-Membrane Interactions using Single Molecule Biophysical Methods.

Mapping membrane protein interactions in cell signaling systems.

We proposed to apply a chemical cross-linking, mass spectrometry and modeling method called MS3D to the structure determination of the rhodopsin-transducin membrane protein complex (RTC). Herein we describe experimental progress made to adapt the MS3D approach for characterizing membrane protein systems, and computational progress in experimental design, data analysis and protein structure modeling. Over the past three years, we have developed tailored experimental methods for all steps in the MS3D method for rhodopsin, including protein purification, a functional assay, cross-linking, proteolysis and mass spectrometry. In support of the experimental effort. we have out a data analysis pipeline in place that automatically selects the monoisotopic peaks in a mass spectrometric spectrum, assigns them and stores the results in a database. Theoretical calculations using 24 experimentally-derived distance constraints have resulted in a backbone-level model of the activated form of rhodopsin, which is a critical first step towards building a model of the RTC. Cross-linked rhodopsin-transducin complexes have been isolated via gel electrophoresis and further mass spectrometric characterization of the cross-links is underway.