data_4937 ####################### # Entry information # ####################### save_entry_information _Saveframe_category entry_information _Entry_title ; 1H chemical shifts for 'Hairpinless' mutant of omega-atracotoxin-Hv1a ; _BMRB_accession_number 4937 _BMRB_flat_file_name bmr4937.str _Entry_type original _Submission_date 2001-01-10 _Accession_date 2001-01-10 _Entry_origination author _NMR_STAR_version 2.1.1 _Experimental_method NMR _Details . loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Fletcher Jamie I. . 2 King Glenn F. . stop_ loop_ _Saveframe_category_type _Saveframe_category_type_count assigned_chemical_shifts 1 stop_ loop_ _Data_type _Data_type_count "1H chemical shifts" 121 stop_ loop_ _Revision_date _Revision_keyword _Revision_author _Revision_detail 2001-01-23 original BMRB . stop_ loop_ _Related_BMRB_accession_number _Relationship 4233 '1H Chemical Shift Assignments for Omega-Atracotoxin-Hv1a' stop_ _Original_release_date 2001-01-10 save_ ############################# # Citation for this entry # ############################# save_entry_citation _Saveframe_category entry_citation _Citation_full . _Citation_title ; Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a ; _Citation_status 'in preparation' _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID ? loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Tedford Hugo W. . 2 Fletcher Jamie I. . 3 King Glenn F. . stop_ _Journal_abbreviation . _Journal_volume . _Journal_issue . _Journal_CSD . _Book_chapter_title . _Book_volume . _Book_series . _Book_ISBN . _Conference_state_province . _Conference_abstract_number . _Page_first . _Page_last . _Year . _Details 'For related publications, see references in saveframes ref_1 and ref_2.' loop_ _Keyword atracotoxin beta-hairpin 'cystine knot' omega-atracotoxin-1 omega-atracotoxin-Hv1a stop_ save_ ####################################### # Cited references within the entry # ####################################### save_Ref_1 _Saveframe_category citation _Citation_full ; 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. ; _Citation_title ; The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider. ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 9228949 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Fletcher J.I. I. . 2 Smith R. . . 3 O'Donoghue S.I. I. . 4 Nilges M. . . 5 Connor M. . . 6 Howden M.E. E. . 7 Christie M.J. J. . 8 King G.F. F. . stop_ _Journal_abbreviation 'Nat. Struct. Biol.' _Journal_name_full 'Nature structural biology' _Journal_volume 4 _Journal_issue 7 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 559 _Page_last 566 _Year 1997 _Details ; A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents. ; save_ save_Ref_2 _Saveframe_category citation _Citation_full ; Wang X, Smith R, Fletcher JI, Wilson H, Wood CJ, Howden ME, King GF. Structure-function studies of omega-atracotoxin, a potent antagonist of insect voltage-gated calcium channels. Eur J Biochem. 1999 Sep;264(2):488-94. ; _Citation_title ; Structure-function studies of omega-atracotoxin, a potent antagonist of insect voltage-gated calcium channels. ; _Citation_status published _Citation_type journal _CAS_abstract_code . _MEDLINE_UI_code . _PubMed_ID 10491095 loop_ _Author_ordinal _Author_family_name _Author_given_name _Author_middle_initials _Author_family_title 1 Wang X. . . 2 Smith R. . . 3 Fletcher J.I. I. . 4 Wilson H. . . 5 Wood C.J. J. . 6 Howden M.E. E. . 7 King G.F. F. . stop_ _Journal_abbreviation 'Eur. J. Biochem.' _Journal_name_full 'European journal of biochemistry / FEBS' _Journal_volume 264 _Journal_issue 2 _Journal_CSD . _Book_title . _Book_chapter_title . _Book_volume . _Book_series . _Book_publisher . _Book_publisher_city . _Book_ISBN . _Conference_title . _Conference_site . _Conference_state_province . _Conference_country . _Conference_start_date . _Conference_end_date . _Conference_abstract_number . _Thesis_institution . _Thesis_institution_city . _Thesis_institution_country . _Page_first 488 _Page_last 494 _Year 1999 _Details ; The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels. ; save_ ################################## # Molecular system description # ################################## save_Hairpinless_omega-ACTX-Hv1a _Saveframe_category molecular_system _Mol_system_name "'Hairpinless' omega-ACTX-Hv1a monomer" _Abbreviation_common 'Hairpinless omega-ACTX-Hv1a' _Enzyme_commission_number . loop_ _Mol_system_component_name _Mol_label 'hairpinless monomer' $hairpinless_monomer stop_ _System_molecular_weight . _System_physical_state native _System_oligomer_state monomer _System_paramagnetic no _System_thiol_state 'all disulfide bound' loop_ _Biological_function 'Hairpinless mutant is inactive' 'Native toxin inhibits insect calcium channels' stop_ _Database_query_date . _Details . save_ ######################## # Monomeric polymers # ######################## save_hairpinless_monomer _Saveframe_category monomeric_polymer _Mol_type polymer _Mol_polymer_class protein _Name_common 'Hairpinless mutant of omega-atracotoxin-Hv1a' _Abbreviation_common Hairpinless _Molecular_mass . _Mol_thiol_state 'all disulfide bound' _Details . ############################## # Polymer residue sequence # ############################## _Residue_count 25 _Mol_residue_sequence ; CIPSGQPCPYNENCCSQSCT GGRCD ; loop_ _Residue_seq_code _Residue_label 1 CYS 2 ILE 3 PRO 4 SER 5 GLY 6 GLN 7 PRO 8 CYS 9 PRO 10 TYR 11 ASN 12 GLU 13 ASN 14 CYS 15 CYS 16 SER 17 GLN 18 SER 19 CYS 20 THR 21 GLY 22 GLY 23 ARG 24 CYS 25 ASP stop_ _Sequence_homology_query_date . _Sequence_homology_query_revised_last_date . save_ #################### # Natural source # #################### save_natural_source _Saveframe_category natural_source loop_ _Mol_label _Organism_name_common _NCBI_taxonomy_ID _Superkingdom _Kingdom _Genus _Species $hairpinless_monomer . 6904 Eukaryota Metazoa Hadronyche versuta stop_ save_ ######################### # Experimental source # ######################### save_experimental_source _Saveframe_category experimental_source loop_ _Mol_label _Production_method _Host_organism_name_common _Genus _Species _Strain _Vector_name $hairpinless_monomer 'chemical synthesis' . . . . . stop_ save_ ##################################### # Sample contents and methodology # ##################################### ######################## # Sample description # ######################## save_Sample_1 _Saveframe_category sample _Sample_type solution _Details . loop_ _Mol_label _Concentration_value _Concentration_value_units _Isotopic_labeling $hairpinless_monomer 3 mM . chloramphenicol 25 uM . TSP 0.1 mM . stop_ save_ ############################ # Computer software used # ############################ save_XWINNMR _Saveframe_category software _Name xwinnmr _Version 2.0 loop_ _Task 'data acquisition' 'data processing' stop_ _Details . save_ save_XEASY _Saveframe_category software _Name XEASY _Version 1.3.13 loop_ _Task 'data acquisition' 'data processing' 'peak integration' 'spectral analysis' stop_ _Details . save_ ######################### # Experimental detail # ######################### ################################## # NMR Spectrometer definitions # ################################## save_NMR_spectrometer _Saveframe_category NMR_spectrometer _Manufacturer Bruker _Model DRX _Field_strength 600 _Details . save_ ############################# # NMR applied experiments # ############################# save_2D_1H-1H_NOESY_1 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H NOESY' _Sample_label $Sample_1 save_ save_2D_1H-1H_TOCSY_2 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H TOCSY' _Sample_label $Sample_1 save_ save_2D_1H-1H_DQF-COSY_3 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H DQF-COSY' _Sample_label $Sample_1 save_ save_2D_1H-1H_E.COSY_4 _Saveframe_category NMR_applied_experiment _Experiment_name '2D 1H-1H E.COSY' _Sample_label $Sample_1 save_ ####################### # Sample conditions # ####################### save_Ex-cond_1 _Saveframe_category sample_conditions _Details . loop_ _Variable_type _Variable_value _Variable_value_error _Variable_value_units 'ionic strength' 0.005 . M pH 4.9 0.05 n/a temperature 298 0.5 K stop_ save_ #################### # NMR parameters # #################### ############################## # Assigned chemical shifts # ############################## ################################ # Chemical shift referencing # ################################ save_chemical_shift_reference _Saveframe_category chemical_shift_reference _Details . loop_ _Mol_common_name _Atom_type _Atom_isotope_number _Atom_group _Chem_shift_units _Chem_shift_value _Reference_method _Reference_type _External_reference_sample_geometry _External_reference_location _External_reference_axis _Indirect_shift_ratio TSP H 1 'methyl protons' ppm 0.00 internal direct . . . 1.0 stop_ save_ ################################### # Assigned chemical shift lists # ################################### ################################################################### # Chemical Shift Ambiguity Index Value Definitions # # # # The values other than 1 are used for those atoms with different # # chemical shifts that cannot be assigned to stereospecific atoms # # or to specific residues or chains. # # # # Index Value Definition # # # # 1 Unique (including isolated methyl protons, # # geminal atoms, and geminal methyl # # groups with identical chemical shifts) # # (e.g. ILE HD11, HD12, HD13 protons) # # 2 Ambiguity of geminal atoms or geminal methyl # # proton groups (e.g. ASP HB2 and HB3 # # protons, LEU CD1 and CD2 carbons, or # # LEU HD11, HD12, HD13 and HD21, HD22, # # HD23 methyl protons) # # 3 Aromatic atoms on opposite sides of # # symmetrical rings (e.g. TYR HE1 and HE2 # # protons) # # 4 Intraresidue ambiguities (e.g. LYS HG and # # HD protons or TRP HZ2 and HZ3 protons) # # 5 Interresidue ambiguities (LYS 12 vs. LYS 27) # # 6 Intermolecular ambiguities (e.g. ASP 31 CA # # in monomer 1 and ASP 31 CA in monomer 2 # # of an asymmetrical homodimer, duplex # # DNA assignments, or other assignments # # that may apply to atoms in one or more # # molecule in the molecular assembly) # # 9 Ambiguous, specific ambiguity not defined # # # ################################################################### save_shift_set_1 _Saveframe_category assigned_chemical_shifts _Details . loop_ _Experiment_label '2D 1H-1H NOESY' '2D 1H-1H TOCSY' '2D 1H-1H DQF-COSY' '2D 1H-1H E.COSY' stop_ loop_ _Sample_label $Sample_1 stop_ _Sample_conditions_label $Ex-cond_1 _Chem_shift_reference_set_label $chemical_shift_reference _Mol_system_component_name 'hairpinless monomer' _Text_data_format . _Text_data . loop_ _Atom_shift_assign_ID _Residue_author_seq_code _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code 1 . 1 CYS HA H 4.58 0.02 1 2 . 1 CYS HB2 H 3.32 0.02 2 3 . 1 CYS HB3 H 3.10 0.02 2 4 . 2 ILE H H 9.25 0.02 1 5 . 2 ILE HA H 4.52 0.02 1 6 . 2 ILE HB H 1.92 0.02 1 7 . 2 ILE HG2 H 1.16 0.02 1 8 . 2 ILE HG12 H 1.70 0.02 2 9 . 2 ILE HD1 H 1.02 0.02 1 10 . 3 PRO HA H 4.49 0.02 1 11 . 3 PRO HB2 H 1.95 0.02 1 12 . 3 PRO HB3 H 2.07 0.02 1 13 . 3 PRO HG2 H 2.43 0.02 1 14 . 3 PRO HG3 H 2.43 0.02 1 15 . 3 PRO HD2 H 4.25 0.02 2 16 . 3 PRO HD3 H 3.64 0.02 2 17 . 4 SER H H 8.53 0.02 1 18 . 4 SER HA H 3.87 0.02 1 19 . 4 SER HB2 H 3.66 0.02 1 20 . 4 SER HB3 H 3.66 0.02 1 21 . 5 GLY H H 9.70 0.02 1 22 . 5 GLY HA2 H 4.42 0.02 2 23 . 5 GLY HA3 H 3.68 0.02 2 24 . 6 GLN H H 7.42 0.02 1 25 . 6 GLN HA H 4.74 0.02 1 26 . 6 GLN HB2 H 2.19 0.02 1 27 . 6 GLN HB3 H 2.04 0.02 1 28 . 6 GLN HG2 H 2.37 0.02 2 29 . 6 GLN HG3 H 2.26 0.02 2 30 . 7 PRO HA H 4.97 0.02 1 31 . 7 PRO HB2 H 1.80 0.02 1 32 . 7 PRO HB3 H 2.15 0.02 1 33 . 7 PRO HG2 H 2.38 0.02 2 34 . 7 PRO HG3 H 1.92 0.02 2 35 . 7 PRO HD2 H 3.84 0.02 2 36 . 7 PRO HD3 H 3.64 0.02 2 37 . 8 CYS H H 8.08 0.02 1 38 . 8 CYS HA H 5.05 0.02 1 39 . 8 CYS HB2 H 3.28 0.02 2 40 . 8 CYS HB3 H 2.76 0.02 2 41 . 9 PRO HA H 4.44 0.02 1 42 . 9 PRO HB2 H 1.98 0.02 1 43 . 9 PRO HB3 H 2.02 0.02 1 44 . 9 PRO HG2 H 1.76 0.02 2 45 . 9 PRO HG3 H 1.57 0.02 2 46 . 9 PRO HD2 H 3.68 0.02 2 47 . 9 PRO HD3 H 3.47 0.02 2 48 . 10 TYR HA H 3.79 0.02 1 49 . 10 TYR HB2 H 2.54 0.02 2 50 . 10 TYR HB3 H 2.16 0.02 2 51 . 10 TYR HD1 H 7.18 0.02 1 52 . 10 TYR HD2 H 7.18 0.02 1 53 . 10 TYR HE1 H 6.81 0.02 1 54 . 10 TYR HE2 H 6.81 0.02 1 55 . 11 ASN H H 8.83 0.02 1 56 . 11 ASN HA H 4.24 0.02 1 57 . 11 ASN HB2 H 2.94 0.02 2 58 . 11 ASN HB3 H 2.77 0.02 2 59 . 11 ASN HD21 H 7.84 0.02 2 60 . 11 ASN HD22 H 7.00 0.02 2 61 . 12 GLU HA H 4.20 0.02 1 62 . 12 GLU HB2 H 2.10 0.02 2 63 . 12 GLU HB3 H 2.02 0.02 2 64 . 12 GLU HG2 H 2.43 0.02 2 65 . 12 GLU HG3 H 2.31 0.02 2 66 . 13 ASN H H 7.71 0.02 1 67 . 13 ASN HA H 4.71 0.02 1 68 . 13 ASN HB2 H 3.08 0.02 2 69 . 13 ASN HB3 H 2.52 0.02 2 70 . 13 ASN HD21 H 7.45 0.02 2 71 . 13 ASN HD22 H 7.00 0.02 2 72 . 14 CYS H H 8.09 0.02 1 73 . 14 CYS HA H 5.02 0.02 1 74 . 14 CYS HB2 H 3.48 0.02 1 75 . 14 CYS HB3 H 2.71 0.02 1 76 . 15 CYS H H 9.85 0.02 1 77 . 15 CYS HA H 4.54 0.02 1 78 . 15 CYS HB2 H 3.31 0.02 1 79 . 15 CYS HB3 H 3.00 0.02 1 80 . 16 SER H H 9.23 0.02 1 81 . 16 SER HA H 4.21 0.02 1 82 . 16 SER HB2 H 4.01 0.02 1 83 . 16 SER HB3 H 3.79 0.02 1 84 . 17 GLN H H 7.77 0.02 1 85 . 17 GLN HA H 3.99 0.02 1 86 . 17 GLN HB2 H 2.34 0.02 2 87 . 17 GLN HB3 H 2.23 0.02 2 88 . 18 SER H H 7.22 0.02 1 89 . 18 SER HA H 4.49 0.02 1 90 . 18 SER HB2 H 3.74 0.02 2 91 . 18 SER HB3 H 3.37 0.02 2 92 . 19 CYS H H 9.10 0.02 1 93 . 19 CYS HA H 4.94 0.02 1 94 . 19 CYS HB2 H 2.93 0.02 1 95 . 19 CYS HB3 H 2.93 0.02 1 96 . 20 THR H H 8.50 0.02 1 97 . 20 THR HA H 4.57 0.02 1 98 . 20 THR HB H 4.18 0.02 1 99 . 20 THR HG2 H 1.20 0.02 1 100 . 21 GLY H H 9.38 0.02 1 101 . 21 GLY HA2 H 3.90 0.02 2 102 . 21 GLY HA3 H 3.82 0.02 2 103 . 22 GLY H H 8.42 0.02 1 104 . 22 GLY HA2 H 4.09 0.02 2 105 . 22 GLY HA3 H 3.79 0.02 2 106 . 23 ARG H H 7.71 0.02 1 107 . 23 ARG HA H 4.96 0.02 1 108 . 23 ARG HB2 H 1.57 0.02 2 109 . 23 ARG HB3 H 1.47 0.02 2 110 . 23 ARG HG3 H 1.37 0.02 2 111 . 23 ARG HD2 H 3.10 0.02 1 112 . 23 ARG HD3 H 3.10 0.02 1 113 . 23 ARG HE H 7.17 0.02 1 114 . 24 CYS H H 8.95 0.02 1 115 . 24 CYS HA H 5.12 0.02 1 116 . 24 CYS HB2 H 3.47 0.02 1 117 . 24 CYS HB3 H 2.94 0.02 1 118 . 25 ASP H H 9.34 0.02 1 119 . 25 ASP HA H 4.41 0.02 1 120 . 25 ASP HB2 H 2.81 0.02 1 121 . 25 ASP HB3 H 2.46 0.02 1 stop_ save_