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Structure and function of ion channel toxins
475
Abstracts Sfructure
381 Royal
nnd,function
Parade,
@ion
channel
Parkville
R. S. Norton
toxins.
(NMR
Laboratory,
Biomolecular
Research
Institute.
3052, Australia).
Ion channel toxins are often small, disulfide-cross-linked proteins with favourable solution properties, the structure determination of which by nuclear magnetic resonance (NMR) is generally straightforward. In this presentation the structures of two polypeptide toxins, both blockers of voltage-gated ion channels, will be described and their mode of interaction with their target channels will be discussed. The first is o-conotoxin GVIA (Pallaghy r( al., 1993), which blocks N-type calcium channels and is of therapeutic interest for its effects on pain perception and as a neuroprotectant in cerebral ischaemia. The structure consists of a small triple-stranded p-sheet and a disulfide knot, which represents a structural motif found in a number of unrelated polypeptides (Pallaghy et al., 1994). An alanine scan has identified some of the residues which contribute to calcium channel binding (Kim et al., 1994); these will be discussed in relation to the likely channel binding surface of the molecule. The second is a novel potassium channel toxin. ShK toxin, isolated from the sea anemone .Stichodac/yla helianthus (Castaiieda ef al., 1995). ShK toxin competes with dendrotoxin-I in binding to rat brain synaptosomes but it is also a potent blocker of Kvl.3 potassium channels in Jurkat T-lymphocytes. As the Kvl.3 channel is implicated in T-lymphocyte proliferation and lymphokine production, blockers of this channel are of interest as potential immunosuppressants. ShK toxin shows little sequence similarity to other potassium channel blockers and has a different disulfide pairing. Our NMR-based structure (Tudor ef al., 1996) confirms that its three-dimensional structure is also different and provides the basis for mapping the potassium channel binding surface(s) of this toxin. A model of how ShK toxin might interact with the potassium channel will be presented. Castaiieda, 0. rf al. (1995) To.vicon 33, 6033613. Kim, J. 1. et al. (1994) J. biol. Chem. 269, 23,876-23.878. Pallaghy, P. K. rf al. (1993) J. Molec. Biol. 234, 405420. Pallaghy, P. K. et al. (1994) Protein Sci. 3, 183331839. Tudor, J. E. et al. (1996) Nature Sir. Biol., in press. S/ructure,,/imction
and A. Menez Fi-ante).
and genetic
(Department
engineering
d’lngenierie
q/ nnimal
et d’Etudes
to.~i-iru. F. Ducancel.
des Proteins.
C. Vita, J.-C. Boulain. S. Zinn-Justin CEA Saclay. 91191 Gif-stir-Yvette Cedex,
Venomous animals are widespread in most phyla of the animal kingdom. They feed on a variety of prey and correspondingly elaborate a variety of toxins. Animal toxins frequently affect the function of ion channels. Most animal toxins are proteins with a small size and a high density of disulfides, and hence with a high stability toward protein denaturants. The three-dimensional structures of several functionally different toxins have been elucidated, by nuclear magnetic resonance spectroscopy and/or X-ray crystallography. These studies have revealed that toxins with different functions can adopt a similar overall conformation. This principle of functional prodigality associated with a structural economy is particularly clear in the cases of scorpions and snakes. Several topographical determinants associated with the capacity of various toxins to recognize their targets have been identified by various approaches, including mutational analyses and structural analyses of toxin-target complexes. These data strongly suggest that any accessible area of a toxin scaffold may potentially become a functional determinant. Interestingly enough, examination of cDNAs and genes encoding snake toxins revealed that the regions coding for the mature part of the toxins undergo an unusually high rate of mutations, suggesting that efficient engineering occurs naturally for these proteins. in venom glands. Based on these observations. a number of toxin scaffolds is now being exploited as templates to engineer novel functions artificially by rational and/or combinatorial approaches, offering new perspectives in the domain of drug design on constrained protein scaffolds. L. Powers. IL R. Sinclair,’ B. Chance’and I. Yamazdki’ (‘National Center Utah State University, Logan. UT 84322-4630. U.S.A.; and ‘University University of Pennsylvania, Philadelphia. PA 19104. U.S.A.).
Molecular design qfpero.yidase function. for the Design of Molecular Function,
of Pennsylvania
Medical
School,
The peroxidase enzyme systems have long been known to demonstrate microbicidal activity and to inactivate bacterial toxins. This activity is related to the formation of peroxidase-derived oxidants. Unlike the oxygen transport hemeproteins, peroxidases are capable of catalyzing the oxidation of substrates with very high redox potentials. such as halide ions and many environmental pollutants. We have compared the structure-function relationship of peroxidases (lignin peroxidases, horseradish peroxidase, lactoperoxidase. myeloperoxidase. cytochrome c peroxidase, Coprinus macrorhizus peroxidase, cataslase. ArthromJces ramosus peroxidase. and microperoxidases MP8 and MPll) with those of other hemeproteins in order to distinguish those molecular design parameters necessary to oxidize high redox potential substrates. The distinguishing features in the active site structures are the proximal H-bonding network, appropriate distal residues for heterolytic cleavage of peroxide, a stable cation radical site, and electron-transfer pathways sufficient for oxidation of substrates. The proximal H-bonding network controls the length of the proximal histidine bond which stabilizes the high valence state intermediates. These intermediates involve the sequential production of a ferry1 iron species and a cation radical which oxidize the substrate in two one-electron steps. The one-electron redox potentials for these intermediate pairs have been measured to be as high as 0.98 V. one of the highest values reported for a biological
Abstracts Sfructure
381 Royal
nnd,function
Parade,
@ion
channel
Parkville
R. S. Norton
toxins.
(NMR
Laboratory,
Biomolecular
Research
Institute.
3052, Australia).
Ion channel toxins are often small, disulfide-cross-linked proteins with favourable solution properties, the structure determination of which by nuclear magnetic resonance (NMR) is generally straightforward. In this presentation the structures of two polypeptide toxins, both blockers of voltage-gated ion channels, will be described and their mode of interaction with their target channels will be discussed. The first is o-conotoxin GVIA (Pallaghy r( al., 1993), which blocks N-type calcium channels and is of therapeutic interest for its effects on pain perception and as a neuroprotectant in cerebral ischaemia. The structure consists of a small triple-stranded p-sheet and a disulfide knot, which represents a structural motif found in a number of unrelated polypeptides (Pallaghy et al., 1994). An alanine scan has identified some of the residues which contribute to calcium channel binding (Kim et al., 1994); these will be discussed in relation to the likely channel binding surface of the molecule. The second is a novel potassium channel toxin. ShK toxin, isolated from the sea anemone .Stichodac/yla helianthus (Castaiieda ef al., 1995). ShK toxin competes with dendrotoxin-I in binding to rat brain synaptosomes but it is also a potent blocker of Kvl.3 potassium channels in Jurkat T-lymphocytes. As the Kvl.3 channel is implicated in T-lymphocyte proliferation and lymphokine production, blockers of this channel are of interest as potential immunosuppressants. ShK toxin shows little sequence similarity to other potassium channel blockers and has a different disulfide pairing. Our NMR-based structure (Tudor ef al., 1996) confirms that its three-dimensional structure is also different and provides the basis for mapping the potassium channel binding surface(s) of this toxin. A model of how ShK toxin might interact with the potassium channel will be presented. Castaiieda, 0. rf al. (1995) To.vicon 33, 6033613. Kim, J. 1. et al. (1994) J. biol. Chem. 269, 23,876-23.878. Pallaghy, P. K. rf al. (1993) J. Molec. Biol. 234, 405420. Pallaghy, P. K. et al. (1994) Protein Sci. 3, 183331839. Tudor, J. E. et al. (1996) Nature Sir. Biol., in press. S/ructure,,/imction
and A. Menez Fi-ante).
and genetic
(Department
engineering
d’lngenierie
q/ nnimal
et d’Etudes
to.~i-iru. F. Ducancel.
des Proteins.
C. Vita, J.-C. Boulain. S. Zinn-Justin CEA Saclay. 91191 Gif-stir-Yvette Cedex,
Venomous animals are widespread in most phyla of the animal kingdom. They feed on a variety of prey and correspondingly elaborate a variety of toxins. Animal toxins frequently affect the function of ion channels. Most animal toxins are proteins with a small size and a high density of disulfides, and hence with a high stability toward protein denaturants. The three-dimensional structures of several functionally different toxins have been elucidated, by nuclear magnetic resonance spectroscopy and/or X-ray crystallography. These studies have revealed that toxins with different functions can adopt a similar overall conformation. This principle of functional prodigality associated with a structural economy is particularly clear in the cases of scorpions and snakes. Several topographical determinants associated with the capacity of various toxins to recognize their targets have been identified by various approaches, including mutational analyses and structural analyses of toxin-target complexes. These data strongly suggest that any accessible area of a toxin scaffold may potentially become a functional determinant. Interestingly enough, examination of cDNAs and genes encoding snake toxins revealed that the regions coding for the mature part of the toxins undergo an unusually high rate of mutations, suggesting that efficient engineering occurs naturally for these proteins. in venom glands. Based on these observations. a number of toxin scaffolds is now being exploited as templates to engineer novel functions artificially by rational and/or combinatorial approaches, offering new perspectives in the domain of drug design on constrained protein scaffolds. L. Powers. IL R. Sinclair,’ B. Chance’and I. Yamazdki’ (‘National Center Utah State University, Logan. UT 84322-4630. U.S.A.; and ‘University University of Pennsylvania, Philadelphia. PA 19104. U.S.A.).
Molecular design qfpero.yidase function. for the Design of Molecular Function,
of Pennsylvania
Medical
School,
The peroxidase enzyme systems have long been known to demonstrate microbicidal activity and to inactivate bacterial toxins. This activity is related to the formation of peroxidase-derived oxidants. Unlike the oxygen transport hemeproteins, peroxidases are capable of catalyzing the oxidation of substrates with very high redox potentials. such as halide ions and many environmental pollutants. We have compared the structure-function relationship of peroxidases (lignin peroxidases, horseradish peroxidase, lactoperoxidase. myeloperoxidase. cytochrome c peroxidase, Coprinus macrorhizus peroxidase, cataslase. ArthromJces ramosus peroxidase. and microperoxidases MP8 and MPll) with those of other hemeproteins in order to distinguish those molecular design parameters necessary to oxidize high redox potential substrates. The distinguishing features in the active site structures are the proximal H-bonding network, appropriate distal residues for heterolytic cleavage of peroxide, a stable cation radical site, and electron-transfer pathways sufficient for oxidation of substrates. The proximal H-bonding network controls the length of the proximal histidine bond which stabilizes the high valence state intermediates. These intermediates involve the sequential production of a ferry1 iron species and a cation radical which oxidize the substrate in two one-electron steps. The one-electron redox potentials for these intermediate pairs have been measured to be as high as 0.98 V. one of the highest values reported for a biological