Three-Dimensional Solution Structure of α-Conotoxin MII, an α3β2 Neuronal Nicotinic Acetylcholine Receptor-Targeted Ligand,

Kj, Shon; Sc, Koerber; Je, Rivier; Olivera, B.M.; McIntosh, J. M.

doi:10.1021/bi971443r

Cited by 55 publications

(61 citation statements)

References 46 publications

(79 reference statements)

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“…There are no solution structures reported for these molecules, but ImI has the same first loop as EpI, and four independent solution structures of ImI have been reported. An N-terminal 3 10 helical motif is reported in just one of the four ImI NMR structures (16), and no other NMR structures of ␣-conotoxins report this element of secondary structure (9,(11)(12)(13)(14)(15)17). In the structure described here a 3 10 helix is detected in 6 of 20 AuIB structures.…”

Section: Fig 7 Effect Of Native and Ribbon Isomers Of Auib On Nicotmentioning

confidence: 99%

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

Dutton

Bansal

Hogg

et al. 2002

Journal of Biological Chemistry

115

129

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␣-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

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Section: Fig 7 Effect Of Native and Ribbon Isomers Of Auib On Nicotmentioning

confidence: 99%

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

Dutton

Bansal

Hogg

et al. 2002

Journal of Biological Chemistry

115

129

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“…The 3D structure of MII consists of a central segment of ␣-helix with ␤-turns at the N and C termini (9,10) and is stabilized by two disulfide bonds in a CysI-CysIII and CysII-CysIV configuration that is common to most members of the ␣-CTX family. In addition, the N and C termini of the peptide are in close proximity to each other, making MII a good candidate for studying the principles of backbone cyclization.…”

mentioning

confidence: 99%

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Clark

Fischer

Dempster

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

212

222

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Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the ␣-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.conotoxins ͉ drug delivery ͉ molecular engineering V enoms from marine snails of the Conus genus comprise a myriad of peptides called conotoxins (CTXs) for the rapid immobilization of prey (1, 2). These 12-to 30-aa peptides target membrane receptors with exquisite selectivity and potency and have become invaluable neurophysiological probes and drug leads. Recently, the CTX ziconotide (MVIIA) was approved for use in the treatment of severe chronic pain by the FDA, and other CTXs have entered clinical trials as treatments for pain (3,4). In addition, CTXs have played a critical role in dissecting the molecular mechanisms of ion channel and transporter functions in the nervous system (2). One family of CTXs, the ␣-CTXs, consists of members that antagonize the nicotinic acetylcholine receptors (nAChRs). Ranging in size from 12 to 19 residues, ␣-CTXs are the smallest of all of the CTXs, yet this family is the most widely distributed among Conus venoms (5).Despite their exciting applications, many peptide toxins are susceptible to enzymatic degradation by proteases. This characteristic may limit the therapeutic applications of CTXs, and, hence, methods that provide improvements in biological half-life are valuable. Cyclization has been used in the past as a strategy in the pharmaceutical industry for stabilizing and locking the conformation of small peptides (6). Similarly, microorganisms are known to produce cyclized peptides, such as cyclosporin A, which is now in widespread use as an immunosuppressant. Such a strategy has not been applied in the past to disulfide-rich proteins, but with the recent discovery of the cyclotide family of macrocyclic miniproteins (7), it is clear that the approach can be applied to disulfide-rich toxins to produce additional stabilization with the potential to dramatically increase the therapeutic potential of these molecules when limited by poor in vivo stability.This stu...

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“…The ␣4/3-type characterized by ImI is ␣7-selective (10). The threedimensional structures of different neuronal-specific and muscle-specific ␣-conotoxins have been determined by NMR (11)(12)(13)(14)(15)) and x-ray structural analysis (16 -19). The ␣4/7-conotoxins and ImI share similar backbone conformations and a rigid hydrophobic core (14), suggesting that their different specificities for nAChR subtypes arise from the different amino acid side-chains projecting from this conserved scaffold.…”

mentioning

confidence: 99%

Isolation, Structure, and Activity of GID, a Novel α4/7-Conotoxin with an Extended N-terminal Sequence

Nicke¹,

Loughnan²,

Millard³

et al. 2003

Journal of Biological Chemistry

127

152

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Using assay-directed fractionation of Conus geographus crude venom, we isolated ␣-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective ␣-conotoxins, ␣-GID has a four amino acid N-terminal tail, ␥-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits ␣7 and ␣3␤2 nAChRs with IC 50 values of 5 and 3 nM, respectively and is at least 1000-fold less potent at the ␣1␤1␥␦, ␣3␤4, and ␣4␤4 combinations. GID also potently inhibits the ␣4␤2 subtype (IC 50 of 150 nM). Deletion of the N-terminal sequence (GID⌬1-4) significantly decreased activity at the ␣4␤2 nAChR but hardly affected potency at ␣3␤2 and ␣7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg 12 contributed to ␣4␤2 and ␣7 activity but not to ␣3␤2 activity. The threedimensional structure of GID is well defined over residues 4 -19 with a similar motif to other ␣-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other ␣4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater ␣7 versus ␣3␤2 selectivity. Neuronal nicotinic acetylcholine receptors (nAChR)1 represent important targets for the development of novel drugs for the treatment of pain and various disorders of the central nervous system (1). To date, eight ␣ and three ␤ subunits (␣2-␣7, ␣9, ␣10, ␤2-␤4) of the nAChRs have been cloned from sensory and neuronal mammalian cells (2-4). For the ␣7 and ␣9 subunits, it has been shown that they need no additional subunits to form functional ion channels upon heterologous expression. All other ␣ subunits, however, require at least the co-expression of one ␤ subunit, or another ␣ subunit in the case of ␣10. Ternary combinations of two different ␣ and one ␤ subunit or two different ␤ and one ␣ subunit, and even quaternary combinations have been described (5, 6). This diversity of subunit combinations has the potential to generate a wide range of receptor subtypes with different pharmacological and functional properties. To help unravel which native neuronal nAChR subunit combinations are responsible for specific physiological functions, additional selective inhibitors are required.Conotoxins are small disulfide-rich peptides from the venom of the predatory marine snails of the genus Conus. These miniproteins have proved to be valuable tools for investigating the structure and function of ligand-and voltage-gated ion channels. ␣-Conotoxins are competitive antagonists of acetylcholine (ACh) binding to the nAChR (7). The ␣-conotoxins described so far are among the most selective inhibitors to be identified (Fig.

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Three-Dimensional Solution Structure of α-Conotoxin MII, an α₃β₂ Neuronal Nicotinic Acetylcholine Receptor-Targeted Ligand^,

Cited by 55 publications

References 46 publications

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Isolation, Structure, and Activity of GID, a Novel α4/7-Conotoxin with an Extended N-terminal Sequence

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