title: Mammalian neuronal sodium channel blocker μ-conotoxin BuIIIB has a structured N-terminus that influences potency.
abstract: Among the μ-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, μ-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact. The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23, and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in μ-BuIIIB, which is also present in μ-BuIIIA and μ-BuIIIC but absent in other μ-conotoxins, forms part of a short α-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related μ-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of μ-BuIIIB for NaV1.3. One analogue, [d-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. On the basis of previous results for related μ-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated and suggest that further gains in potency might be achieved by additional modifications of this region.
authors.affiliation: The Walter and Eliza Hall Institute of Medical Research , 1G Royal Parade, Parkville, Victoria, 3052, Australia.
authors.forename: Zhihe
authors.initials: Z
authors.lastname: Kuang
authors.forename: Min-Min
authors.initials: MM
authors.lastname: Zhang
authors.forename: Kallol
authors.initials: K
authors.lastname: Gupta
authors.forename: Joanna
authors.initials: J
authors.lastname: Gajewiak
authors.forename: Jozsef
authors.initials: J
authors.lastname: Gulyas
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Balaram
authors.forename: Jean E
authors.initials: JE
authors.lastname: Rivier
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Doju
authors.initials: D
authors.lastname: Yoshikami
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bulaj
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Norton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020916:Conotoxins / Q000737:chemistry* / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D062552:NAV1.3 Voltage-Gated Sodium Channel / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000737:chemistry* / Q000494:pharmacology*; D014981:Xenopus
chemical_list: D020916:Conotoxins; D004220:Disulfides; D062552:NAV1.3 Voltage-Gated Sodium Channel; D026941:Sodium Channel Blockers
references.citation: Channels (Austin). 2009 Jan-Feb;3(1):32-8
references.pmid: 19221510
references.citation: Biochemistry. 2008 Oct 14;47(41):10940-9
references.pmid: 18798648
references.citation: Biochemistry. 2010 Jun 15;49(23):4804-12
references.pmid: 20459109
references.citation: Biopolymers. 1968 Oct;6(10):1425-36
references.pmid: 5685102
references.citation: J Mol Biol. 1993 Nov 20;234(2):405-20
references.pmid: 8230223
references.citation: Toxicon. 2006 Dec 1;48(7):780-98
references.pmid: 16952384
references.citation: J Med Chem. 2011 Nov 10;54(21):7558-66
references.pmid: 21962108
references.citation: J Neurosci. 1998 Jun 15;18(12):4473-81
references.pmid: 9614224
references.citation: Mar Drugs. 2010 Jul 13;8(7):2153-61
references.pmid: 20714429
references.citation: Anal Chem. 2010 Oct 1;82(19):8313-9
references.pmid: 20843009
references.citation: Biochemistry. 2009 Feb 17;48(6):1210-9
references.pmid: 19170536
references.citation: J Biol Chem. 2007 Oct 19;282(42):30699-706
references.pmid: 17724025
references.citation: Mol Pharmacol. 2007 Mar;71(3):676-85
references.pmid: 17142296
references.citation: J Neurosci. 2000 Jan 1;20(1):76-80
references.pmid: 10627583
references.citation: Curr Pharm Des. 2008;14(24):2462-79
references.pmid: 18781995
references.citation: J Biol Chem. 2012 Sep 7;287(37):31382-92
references.pmid: 22773842
references.citation: Br J Pharmacol. 2012 Jul;166(5):1654-68
references.pmid: 22229737
references.citation: J Biol Chem. 2003 Mar 7;278(10):8717-24
references.pmid: 12471026
references.citation: Annu Rev Neurosci. 2010;33:325-47
references.pmid: 20367448
references.citation: Toxicon. 2009 Jan;53(1):90-8
references.pmid: 18950653
references.citation: Nucleic Acids Res. 2008 Jan;36(Database issue):D402-8
references.pmid: 17984079
references.citation: Pharmacol Rev. 2005 Dec;57(4):397-409
references.pmid: 16382098
references.citation: Molecules. 2010 Apr 19;15(4):2825-44
references.pmid: 20428082
references.citation: J Biol Chem. 2002 Jul 26;277(30):27247-55
references.pmid: 12006587
references.citation: Mol Pharmacol. 2011 Oct;80(4):573-84
references.pmid: 21709136
references.citation: Anal Chem. 2012 Jan 3;84(1):262-6
references.pmid: 22126836
references.citation: J Neurophysiol. 2010 Jul;104(1):88-97
references.pmid: 20410356
references.citation: Curr Med Chem. 2004 Dec;11(23):3053-64
references.pmid: 15578999
references.citation: Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10302-7
references.pmid: 21652775
references.citation: Br J Pharmacol. 2012 Oct;167(3):576-86
references.pmid: 22537004
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):235-42
references.pmid: 10592235
references.citation: J Biol Chem. 2003 Nov 21;278(47):46805-13
references.pmid: 12970353
references.citation: Mol Biosyst. 2013 Jun;9(6):1340-50
references.pmid: 23467691
references.citation: Biochemistry. 2012 Dec 11;51(49):9826-35
references.pmid: 23167564
references.citation: Physiol Rev. 2004 Jan;84(1):41-68
references.pmid: 14715910
references.citation: Biochemistry. 2002 Dec 24;41(51):15388-93
references.pmid: 12484778
references.citation: Pain. 2007 Oct;131(3):243-257
references.pmid: 17766042
references.citation: J Biol Chem. 1985 Aug 5;260(16):9280-8
references.pmid: 2410412
references.citation: Chem Biol. 2007 Apr;14(4):399-407
references.pmid: 17462575
references.citation: Biochemistry. 2005 May 17;44(19):7259-65
references.pmid: 15882064