Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ω-conotoxin GVIA and its selenocysteine analogs.
Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Konkallu Hanumae
KH
Gowd
Kirk D
KD
Blais
Keith S
KS
Elmslie
Andrew M
AM
Steiner
Baldomero M
BM
Olivera
Grzegorz
G
Bulaj
D000595:Amino Acid Sequence; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000737:chemistry*; D019418:Evolution, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D059748:Proteolysis; D017279:Selenocysteine / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020866:omega-Conotoxin GVIA / Q000737:chemistry*
D004220:Disulfides; D010455:Peptides; D017279:Selenocysteine; D020866:omega-Conotoxin GVIA; D003545:Cysteine