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| - | [[Image:1hvw.gif|left|200px]]<br /><applet load="1hvw" size="450" color="white" frame="true" align="right" spinBox="true" | |
| - | caption="1hvw" /> | |
| - | '''HAIRPINLESS MUTANT OF OMEGA-ATRACOTOXIN-HV1A'''<br /> | |
| | | | |
| - | ==Overview== | + | ==HAIRPINLESS MUTANT OF OMEGA-ATRACOTOXIN-HV1A== |
| - | omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic, specificity derives from its ability to antagonize insect, but not, vertebrate, voltage-gated calcium channels. In order to help understand, its mechanism of action and to enhance its utility as a lead compound for, insecticide development, we used a combination of protein engineering and, site-directed mutagenesis to probe the toxin for key functional regions., First, we constructed a Hairpinless mutant in which the C-terminal, beta-hairpin, which is highly conserved in this family of neurotoxins, was, excised without affecting the fold of the residual disulfide-rich core of, the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently, developed a highly efficient system for production of recombinant toxin, and then probed the hairpin for key functional residues using, alanine-scanning mutagenesis followed by a second round of mutagenesis, based on initial "hits" from the alanine scan. This revealed that two, spatially proximal residues, Asn(27) and Arg(35), form a contiguous, molecular surface that is essential for toxin activity. We propose that, this surface of the beta-hairpin is a key site for interaction of the, toxin with insect calcium channels. | + | <StructureSection load='1hvw' size='340' side='right'caption='[[1hvw]]' scene=''> |
| | + | == Structural highlights == |
| | + | <table><tr><td colspan='2'>[[1hvw]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Hadronyche_versuta Hadronyche versuta]. Full experimental information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1HVW OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1HVW FirstGlance]. <br> |
| | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">Solution NMR, 20 models</td></tr> |
| | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1hvw FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1hvw OCA], [https://pdbe.org/1hvw PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1hvw RCSB], [https://www.ebi.ac.uk/pdbsum/1hvw PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1hvw ProSAT]</span></td></tr> |
| | + | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/TO1A_HADVE TO1A_HADVE] Reversibly and voltage-independently blocks both mid-low- (M-LVA) and high-voltage-activated (HVA) calcium channels in cockroach DUM neurons (PubMed:17610847). Is lethal to many insect species but not toxic to mammals (PubMed:16779650, PubMed:9228949). May target the insect high-voltage-activated calcium channel Dmca1D. Also inhibits acarines calcium channels. An extremely high toxin concentration partially inhibits Cav1.2/CACNA1C, Cav2.1/CACNA1A and Cav2.2/CACNA1B calcium channel of rats. As for omega-AcTx-Hv2a, the phenotypic effect of injection of this toxin into lone star ticks (Amblyomma americanum) is curling of all eight legs into closed loops.<ref>PMID:14608494</ref> <ref>PMID:15308644</ref> <ref>PMID:16330063</ref> <ref>PMID:17141372</ref> <ref>PMID:17610847</ref> <ref>PMID:9228949</ref> |
| | + | <div style="background-color:#fffaf0;"> |
| | + | == Publication Abstract from PubMed == |
| | + | omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels. |
| | | | |
| - | ==About this Structure==
| + | Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a.,Tedford HW, Fletcher JI, King GF J Biol Chem. 2001 Jul 13;276(28):26568-76. Epub 2001 Apr 19. PMID:11313356<ref>PMID:11313356</ref> |
| - | 1HVW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/ ]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1HVW OCA].
| + | |
| | | | |
| - | ==Reference==
| + | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
| - | Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a., Tedford HW, Fletcher JI, King GF, J Biol Chem. 2001 Jul 13;276(28):26568-76. Epub 2001 Apr 19. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=11313356 11313356]
| + | </div> |
| - | [[Category: Single protein]] | + | <div class="pdbe-citations 1hvw" style="background-color:#fffaf0;"></div> |
| - | [[Category: Fletcher, J.I.]] | + | == References == |
| - | [[Category: King, G.F.]] | + | <references/> |
| - | [[Category: beta-hairpin]] | + | __TOC__ |
| - | [[Category: cystine knot]]
| + | </StructureSection> |
| - | | + | [[Category: Hadronyche versuta]] |
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 16:51:34 2007''
| + | [[Category: Large Structures]] |
| | + | [[Category: Fletcher JI]] |
| | + | [[Category: King GF]] |
| Structural highlights
Function
TO1A_HADVE Reversibly and voltage-independently blocks both mid-low- (M-LVA) and high-voltage-activated (HVA) calcium channels in cockroach DUM neurons (PubMed:17610847). Is lethal to many insect species but not toxic to mammals (PubMed:16779650, PubMed:9228949). May target the insect high-voltage-activated calcium channel Dmca1D. Also inhibits acarines calcium channels. An extremely high toxin concentration partially inhibits Cav1.2/CACNA1C, Cav2.1/CACNA1A and Cav2.2/CACNA1B calcium channel of rats. As for omega-AcTx-Hv2a, the phenotypic effect of injection of this toxin into lone star ticks (Amblyomma americanum) is curling of all eight legs into closed loops.[1] [2] [3] [4] [5] [6]
Publication Abstract from PubMed
omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels.
Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a.,Tedford HW, Fletcher JI, King GF J Biol Chem. 2001 Jul 13;276(28):26568-76. Epub 2001 Apr 19. PMID:11313356[7]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Bloomquist JR. Mode of action of atracotoxin at central and peripheral synapses of insects. Invert Neurosci. 2003 Nov;5(1):45-50. Epub 2003 Nov 8. PMID:14608494 doi:http://dx.doi.org/10.1007/s10158-003-0027-z
- ↑ Tedford HW, Gilles N, Menez A, Doering CJ, Zamponi GW, King GF. Scanning mutagenesis of omega-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels. J Biol Chem. 2004 Oct 15;279(42):44133-40. Epub 2004 Aug 11. PMID:15308644 doi:http://dx.doi.org/10.1074/jbc.M404006200
- ↑ Mukherjee AK, Sollod BL, Wikel SK, King GF. Orally active acaricidal peptide toxins from spider venom. Toxicon. 2006 Feb;47(2):182-7. Epub 2005 Dec 5. PMID:16330063 doi:http://dx.doi.org/10.1016/j.toxicon.2005.10.011
- ↑ Tedford HW, Maggio F, Reenan RA, King G. A model genetic system for testing the in vivo function of peptide toxins. Peptides. 2007 Jan;28(1):51-6. Epub 2006 Dec 1. PMID:17141372 doi:http://dx.doi.org/10.1016/j.peptides.2006.08.026
- ↑ Chong Y, Hayes JL, Sollod B, Wen S, Wilson DT, Hains PG, Hodgson WC, Broady KW, King GF, Nicholson GM. The omega-atracotoxins: selective blockers of insect M-LVA and HVA calcium channels. Biochem Pharmacol. 2007 Aug 15;74(4):623-38. Epub 2007 May 25. PMID:17610847 doi:http://dx.doi.org/10.1016/j.bcp.2007.05.017
- ↑ Fletcher JI, Smith R, O'Donoghue SI, Nilges M, Connor M, Howden ME, Christie MJ, King GF. The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nat Struct Biol. 1997 Jul;4(7):559-66. PMID:9228949
- ↑ Tedford HW, Fletcher JI, King GF. Functional significance of the beta hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a. J Biol Chem. 2001 Jul 13;276(28):26568-76. Epub 2001 Apr 19. PMID:11313356 doi:http://dx.doi.org/10.1074/jbc.M102199200
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