Three-Dimensional Solution Structure of α-Conotoxin MII, an α3β2 Neuronal Nicotinic Acetylcholine Receptor-Targeted Ligand (original) (raw)
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Biochemistry, 1998
The three-dimensional structure of conotoxin ψ-PIIIE, a 24-amino acid peptide from Conus purpurascens, has been solved using two-dimensional (2D) 1 H NMR spectroscopy. Conotoxin ψ-PIIIE contains the same disulfide bonding pattern as the µ-conotoxins, which target skeletal muscle sodium channels, but has been shown to antagonize the acetylcholine gated cation channel through a noncompetitive mechanism. Structural information was obtained by the analysis of a series of 2D NOESY spectra as well as measurement of coupling constants from 1D 1 H and PE-COSY NMR experiments. Molecular modeling calculations included the use of the distance geometry (DG) algorithm, simulated annealing techniques, and the restrained molecular dynamics method. The resulting structures are considerably similar to the previously published structures for the µ-conotoxins GIIIA and GIIIB, despite the lack of sequence conservation between conotoxin ψ-PIIIE and the µ-conotoxins. The structure consists of a series of tight turns, each turn occurring in the position analogous to those of turns described in µ-GIIIA and µ-GIIIB. This suggests the disulfide bonding pattern is able to largely direct the structure of the peptides, creating a stable structural motif which allows extensive sequence substitution of non-cystine residues. † Supported by NIH Grants P01 GM48677 and GM54710. ‡ Coordinates for the final structures have been deposited at the Brookhaven Protein Data Bank, Upton, NY 11973, under accession code 1as5.
Three-dimensional structure of α-conotoxin EI determined by 1 H NMR spectroscopy
International Journal of Peptide Research and Therapeutics
Summary α-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom ofConus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organTorpedo, showing a novel selectivity profile when compared to other α-conotoxins. The D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between THr9 and Met12 that is stabilized by the Cys4-Cys10 disulfide bond and turns involving Cys4-Cys5 and Asn14-Pro15. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7...
Biochemistry, 1999
The three-dimensional solution conformation of an 11-residue antitoxic analogue of R-conotoxin GI, des-Glu1-[Cys3Ala]-des-Cys13-conotoxin GI (CANPACGRHYS-NH 2 , designated "GI-15" henceforth), has been determined using two-dimensional 1 H NMR spectroscopy. The disulfide loop region (1C-6C) and the C-terminal tail (8R-11S) are connected by a flexible hinge formed near 7G, and the pairwise backbone rmsds for the former and the latter are 0.58 and 0.65 Å, respectively. Superpositioning GI-15 with the structure of R-conotoxin GI shows that the two share an essentially identical fold in the common first disulfide loop region (1C-6C). However, the absence of the second disulfide loop in GI-15 results in segmental motion of the C-terminal half, causing the key receptor subtype selectivity residue 8R (Arg9 in R-conotoxin GI) to lose its native spatial orientation. The combined features of structural equivalence in the disulfide loop and a mobile C-terminal tail appear to be responsible for the activity of GI-15 as a competitive antagonist against native toxin. Electrostatic surface potential comparisons of the first disulfide region of GI-15 with other R-conotoxins or receptor-bound states of acetylcholine and d-tubocurarine show a common protruding surface that may serve as the minimal binding determinant for the neuromuscular acetylcholine receptor R 1 -subunit. On the basis of the original "Conus toxin macrosite model" [Olivera, B. FIGURE 3: Stereoviews of the 30 final structures of GI-15 superimposed over the backbone atoms (N, C R , C′, and O) of (a) residues 1-6 and (b) residues 7-11. Residues 1-6 are not shown in panel b. Selected residues are labeled.
Biochemical and Biophysical Research Communications, 2006
We have determined a high-resolution three-dimensional structure of a-conotoxin BuIA, a 13-residue peptide toxin isolated from Conus bullatus. Despite its unusual 4/4 disulfide bond layout a-conotoxin BuIA exhibits strong antagonistic activity at a6/a3b2b3, a3b2, and a3b4 nAChR subtypes like some a4/7 conotoxins. a-Conotoxin BuIA lacks the C-terminal b-turn present within the second disulfide loop of a4/7 conotoxins, having only a ''pseudo x-shaped'' molecular topology. Nevertheless, it contains a functionally critical two-turn helix motif, a feature ubiquitously found in a4/7 conotoxins. Such an aspect seems mainly responsible for similarities in the receptor recognition profile of a-conotoxin BuIA to a4/7 conotoxins. Structural comparison of a-conotoxin BuIA with a4/7 conotoxins and a4/3 conotoxin ImI suggests that presence of the second helical turn portion of the two-turn helix motif in a4/7 and a4/4 conotoxins may be important for binding to the a3 and/or a6 subunit of nAChR.
Biochemistry
The three-dimensional solution conformation of an 11-residue antitoxic analogue of alpha-conotoxin GI, des-Glu1-[Cys3Ala]-des-Cys13-conotoxin GI (CANPACGRHYS-NH(2), designated "GI-15" henceforth), has been determined using two-dimensional (1)H NMR spectroscopy. The disulfide loop region (1C-6C) and the C-terminal tail (8R-11S) are connected by a flexible hinge formed near 7G, and the pairwise backbone rmsds for the former and the latter are 0.58 and 0.65 A, respectively. Superpositioning GI-15 with the structure of alpha-conotoxin GI shows that the two share an essentially identical fold in the common first disulfide loop region (1C-6C). However, the absence of the second disulfide loop in GI-15 results in segmental motion of the C-terminal half, causing the key receptor subtype selectivity residue 8R (Arg9 in alpha-conotoxin GI) to lose its native spatial orientation. The combined features of structural equivalence in the disulfide loop and a mobile C-terminal tail appear...
Structure, 1996
Background: ␣-Conotoxins are peptide toxins, isolated from Conus snails, that block the nicotinic acetylcholine receptor (nAChR). The 16-residue peptides PnIA and PnIB from Conus pennaceus incorporate the same disulfide framework as other ␣-conotoxins but differ in function from most ␣-conotoxins by blocking the neuronal nAChR, rather than the skeletal muscle subtype. The crystal structure determination of PnIA was undertaken to identify structural and surface features that might be important for biological activity.
Journal of Medicinal Chemistry, 2008
R-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized R-conotoxins have a 4/7 loop size, and the major features of neuronal R-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of R-conotoxins. This involved the systematic truncation of the second loop in the R-conotoxin [A10L]PnIA [4/7], a potent antagonist of the R7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at R7 nAChRs, whereas 4/3[A10L]PnIA lost both R7 nAChR activity and helicity. In contrast, all truncated analogues lost ∼100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the R7 nAChR and AChBP.
Journal of Biological Chemistry, 2000
The neuronal nicotinic acetylcholine receptors constitute a highly diverse group, with subtypes consisting of pentameric combinations of ␣ and  subunits. ␣-Conotoxins are a homologous series of small peptides that antagonize these receptors. We present the three-dimensional solution structure of ␣-conotoxin AuIB, the first 15-residue ␣-conotoxin known to selectively block the ␣ 3  4 nicotinic acetylcholine receptor subtype. The pairwise backbone and heavy-atom root mean square deviation for an ensemble of 20 structures are 0.269 and 0.720 Å, respectively. The overall fold of ␣-conotoxin AuIB closely resembles that of the ␣4/7 subfamily ␣-conotoxins. However, the absence of Tyr 15 , normally present in other ␣4/7 members, results in tight bending of the backbone at the C terminus and effectively renders Asp 14 to assume the spatial location of Tyr 15 present in other neuronal ␣4/7 ␣-conotoxins. Structural comparison of ␣-conotoxin AuIB with the ␣ 3  2 subtype-specific ␣-conotoxin MII shows different electrostatic surface charge distributions, which may be important in differential receptor subtype recognition.
Investigation of Conotoxin Binding to Nicotinic Acetylcholine Receptor from Free Energy Calculations
At the present time analgesics (or pain killer) are produced mostly based on Morphine. However, regular use of Morphine can cause strong addiction. They bind to Opioid Receptors as agonists of Endorphins. This fact may result in cutting of the production of Endorphins by the human body. In order to discover an alternative analgesic to morphine, investigation of the interaction mechanism between Nicotinic Acetylcholine Receptors (nAChRs) and Conotoxins plays an important role in the discovery of novel analgesics. nAChRs take place among the most crucial proteins in living bodies. They are responsible for the contraction of muscles by leading Na+ ions to enter into the cells. On the other hand, if some molecule blocks this protein, then it results in paralyses. This fact makes their blockers potential analgesics. For such study it was a challenge that the 3D structure of the nAChR was complex to designate. On the other hand, it has been identified by Unwin in 2006. In this study, we have carried out Molecular Dynamics (MD) simulations to the most probable complex structures. In order to get complex structures we have used Haddock software package. From the data obtained from MD simulations, we have carried out Potential of Mean Force (PMF) calculations using Umbrella Sampling (US) techniques. The PMF data can be compared directly with the experimental results in terms of Free Energies. Since US is a strong technique, our results have been confirmed with regard to the previous experimental studies.