title: Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ.
abstract: Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin μO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin μO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of μO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic μO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel β-strands stabilized by three disulfide bridges. The loop region linking the β-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of μO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of μO§-GVIIJ on a distinct surface of the peptide.
authors.affiliation: From the Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia, the Department of Biology, University of Utah, Salt Lake City, Utah 84112.
authors.forename: Brad R
authors.initials: BR
authors.lastname: Green
authors.affiliation: the Department of Biology, University of Utah, Salt Lake City, Utah 84112.
authors.forename: Joanna
authors.initials: J
authors.lastname: Gajewiak
authors.affiliation: From the Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Chhabra
authors.affiliation: the Department of Biochemistry and.
authors.forename: Jack J
authors.initials: JJ
authors.lastname: Skalicky
authors.affiliation: the Department of Biology, University of Utah, Salt Lake City, Utah 84112.
authors.forename: Min-Min
authors.initials: MM
authors.lastname: Zhang
authors.affiliation: the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037.
authors.forename: Jean E
authors.initials: JE
authors.lastname: Rivier
authors.affiliation: the Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108, and.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bulaj
authors.affiliation: the Department of Biology, University of Utah, Salt Lake City, Utah 84112.
authors.forename: Baldomero M
authors.identifier: http://orcid.org/0000-0003-4556-1410
authors.initials: BM
authors.lastname: Olivera
authors.affiliation: the Department of Biology, University of Utah, Salt Lake City, Utah 84112, yoshikami@bioscience.utah.edu.
authors.forename: Doju
authors.initials: D
authors.lastname: Yoshikami
authors.affiliation: From the Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia, ray.norton@monash.edu.
authors.forename: Raymond S
authors.identifier: http://orcid.org/0000-0001-8893-0584
authors.initials: RS
authors.lastname: Norton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020916:Conotoxins / Q000138:chemical synthesis / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012908:Snails / Q000737:chemistry; D026941:Sodium Channel Blockers / Q000138:chemical synthesis / Q000737:chemistry*; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D060327:Solid-Phase Synthesis Techniques; D013329:Structure-Activity Relationship
chemical_list: D020916:Conotoxins; D004220:Disulfides; D009124:Muscle Proteins; D062551:NAV1.2 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C466180:Scn4a protein, rat; D026941:Sodium Channel Blockers; D015222:Sodium Channels
keywords: conotoxin; cysteine; disulfide; high performance liquid chromatography (HPLC); nuclear magnetic resonance (NMR); peptide chemical synthesis; sodium channel; structure-activity relationship studies; two-electrode voltage clamp electrophysiology
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