The Metal-free and Calcium-bound Structures of a γ-Carboxyglutamic Acid-containing Contryphan from Conus marmoreus, Glacontryphan-M
2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês
10.1074/jbc.m313826200
ISSN1083-351X
AutoresMarianne A. Grant, Karin Hansson, Barbara C. Furie, Bruce Furie, Johan Stenflo, Alan C. Rigby,
Tópico(s)Receptor Mechanisms and Signaling
ResumoGlacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca2+ channels expressed in mouse pancreatic β-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453–32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a d-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two γ-carboxyglutamic acid (Gla) residues, formed by post-translational modification of glutamic acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional 1H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys11-Cys5-Pro6 region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla2 and Ser3, moves away from the His8 and Trp10 aromatic rings and the alignment of the d-Trp7 and His8 aromatic rings with respect to the Trp10 rings is altered. The blockage of L-type voltage-gated Ca2+ channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone Cα conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 Å, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation. Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca2+ channels expressed in mouse pancreatic β-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453–32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a d-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two γ-carboxyglutamic acid (Gla) residues, formed by post-translational modification of glutamic acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional 1H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys11-Cys5-Pro6 region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla2 and Ser3, moves away from the His8 and Trp10 aromatic rings and the alignment of the d-Trp7 and His8 aromatic rings with respect to the Trp10 rings is altered. The blockage of L-type voltage-gated Ca2+ channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone Cα conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 Å, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation. Venomous marine snails belonging to the genus Conus synthesize neuropharmacologically active peptides, conotoxins, which demonstrate unique functional properties for use in prey envenomation and self-defense mechanisms. Conotoxins behave as antagonists, exhibiting molecular specificity for receptor isoforms and ion channels in the neuromuscular system. Most conotoxins appear to be derived from a few gene superfamilies, each distinguished by conserved signal and precursor sequence elements (1Espiritu D.J.D. Watkins M. Dia-Monje V. Cartier G.E. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 1899-1916Google Scholar). The hallmark of these conotoxins is their conformationally constrained structure, often created through disulfide bridges. These conserved structural scaffolds provide a framework for the presentation of intervening hypervariable regions comprised of post-translationally modified amino acids that contribute to molecular specificity. Conotoxins of the contryphan family have been found with post-translational modifications including proline hydroxylation, C-terminal amidation, tryptophan bromination (2Jimenez E.C. Craig A.G. Watkins M. Hillyard D.R. Gray W.R. Gulyas J. Rivier J.E. Cruz L.J. Olivera B.M. Biochemistry. 1997; 36: 989-994Google Scholar), and leucine and tryptophan epimerization (3Jimenez E.C. Olivera B.M. Gray W.R. Cruz L.J. J. Biol. Chem. 1996; 271: 28002-28005Google Scholar, 4Jacobsen R.B. Jimenez E.C. De la Cruz R.G.C. Gray W.R. Cruz L.J. Olivera B.M. J. Peptide Res. 1999; 54: 93-99Google Scholar). The nine contryphans characterized to date share the conserved sequence motif, CP*(d-W or d-L)XPWC, that includes a tryptophan or leucine in the d-conformation, a disulfide bond between the two cysteines, and in some cases hydroxylation of the proline preceding the d-Trp residue, Pro*. The N-terminal Cys-Pro peptide bond exhibits cis-trans isomerization in the contryphans characterized to date; however, the more abundant cis isomer is believed to be the functionally relevant conformer (5Pallaghy P.K. He W. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 2000; 39: 12845-12852Google Scholar). The contryphan motif CP*(d-W or L)XPWC is a robust structural scaffold, that maintains the backbone structure and α-β bond vector orientation independent of the amino acid that is substituted at residue X (6Pallaghy P.K. Norton R.S. Biopolymers. 2000; 54: 173-179Google Scholar). This residue is Gln in contryphan-Sm (5Pallaghy P.K. He W. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 2000; 39: 12845-12852Google Scholar), Glu in contryphan-R (7Pallaghy P.K. Melnikova A.P. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 1999; 28: 11553-11559Google Scholar), and Lys in contryphan-Vn (8Massilia G.R. Schinina M.E. Ascenzi P. Polticelli F. Biochem. Biophys. Res. Commun. 2001; 288: 908-913Google Scholar). Functionally, the contryphans elicit a stiff tail syndrome in mice when injected intracranially, or a body tremor and mucous secretion when injected intramuscularly into fish (9Jimenez E.C. Watkins M. Juszczak L.J. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 803-808Google Scholar). Recently, contryphan-Vn from Conus ventricosus has been shown to affect both voltage-gated and Ca2+-dependent potassium channels (K+ channels) (10Massilia G.R. Eliseo T. Grolleau F. Lapied B. Barbier J. Bournaud R. Molgo J. Cicero D.O. Paci M. Schinina M.E. Ascenzi P. Polticelli F. Biochem. Biophys. Res. Commun. 2003; 303: 238-246Google Scholar), identifying the first functional target for a contryphan. In the accompanying article (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar) we describe the purification, primary structure, and functional characterization of a novel contryphan from the venom of Conus marmoreous, glacontryphan-M (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar). Glacontryphan-M, with the sequence NγSγCP(d-W)HPWC-NH2 (γ-carboxyglutamic acid (Gla) 1The abbreviations used are: Gla, γ-carboxyglutamic acid; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root mean-squared deviation. 1The abbreviations used are: Gla, γ-carboxyglutamic acid; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root mean-squared deviation. residue; NH2, C-terminal amidation), is the first contryphan found to contain Gla, a post-translationally modified glutamic acid residue (Table I). The malonate-like side chain of Gla can chelate divalent metal ions to support metal ion-dependent structural determinants that are critical for function.Table 1Comparison of the sequences of contryphans with known NMR structuresConopeptideAmino acid sequenceaO, 4-trans-hydroxyproline; W, D-tryptophan, γ, gamma-carboxyglutamic acid.,bThe hypervariable residue within the intercysteine-loop is in bold.2SpeciesRef.Contryphan-RGCOWEPWC-NH2C. radiatusJiminez et al., 1996Contryphan-SmCOWQPWC-NH2C. stercusmuscarumJacobsen et al., 1998Contryphan-VnGDCPWKPWC-NH2C. ventricosusMassilia et al., 2001Glacontryphan-MNγSγCPWHPWC-NH2C. marmoreusHansson et al., 2004a O, 4-trans-hydroxyproline; W, D-tryptophan, γ, gamma-carboxyglutamic acid.b The hypervariable residue within the intercysteine-loop is in bold. Open table in a new tab To date many Gla-containing conotoxins have been identified and characterized (12McIntosh J.M. Olivera B.M. Cruz L.J. Gray W.R. J. Biol. Chem. 1984; 259: 14343-14346Google Scholar, 13Haack J.A. Rivier J. Parks T.N. Mena E.E. Cruz L.J. Olivera B.M. J. Biol. Chem. 1990; 265: 6025-6029Google Scholar, 14Nakamura T. Yu Z. Fainzilber M. Burlingame A.L. Protein Sci. 1996; 5: 524-530Google Scholar). The best studied Gla-containing conotoxin is conanotokin-G, a 17-residue neuroactive peptide from Conus geographus containing five Gla residues, which antagonizes specific isoforms of the N-methyl-d-aspartate (NMDA) receptor (12McIntosh J.M. Olivera B.M. Cruz L.J. Gray W.R. J. Biol. Chem. 1984; 259: 14343-14346Google Scholar). The metal binding properties and three-dimensional structure of conantokin-G suggest a structural role for Gla, whereby calcium ion binding by Gla residues increases the α-helicity and structural rigidity of the conantokin (16Rigby A.C. Baleja J.D. Li L. Pedersen L.G. Furie B.C. Furie B. Biochemistry. 1997; 36: 15677-15684Google Scholar, 17Skjaerbaek N. Nielsen K.J. Lewis J. Alewood P. Craik D.J. J. Biol. Chem. 1997; 272: 2291-2299Google Scholar, 18Blandl T. Warder S.E. Prorok M. Castellino F.J. J. Peptide. Res. 1999; 53: 453-464Google Scholar). Another protein family rich in Gla and functionally dependent on divalent metal ion interactions is the vitamin K-dependent protein family, including factor IX, factor VII, prothrombin, and factor X that participate in blood coagulation in vertebrates (Refs. 19Furie B. Furie B.C. Cell. 1988; 53: 505-518Google Scholar and 20Stenflo J. Dahlback B. Stamatoyannopoulus G. Majerus P.W. Perlmutter R.M. Varmus H. The Molecular Basis of Blood Disease. WB Saunders, New York2001: 579-613Google Scholar and references therein). It is well established that the vitamin K-dependent proteins bind to negatively charged phospholipids through a mechanism involving the N-terminal Gla domain (21Sunnerhagen M. Forsen S. Hoffren A.-M. Drakenberg T. Teleman O. Stenflo J. Nat. Struct. Biol. 1995; 2: 504-509Google Scholar, 22Freedman S.J. Blostein M.D Baleja J.D. Jacobs M. Furie B.C. Furie B. J. Biol. Chem. 1996; 271: 16227-16236Google Scholar, 23Huang M. Rigby A.C. Morelli X. Grant M.A. Huang G. Furie B. Seaton B. Furie B.C. Nat. Struct. Biol. 2003; 10: 751-756Google Scholar). Nine to twelve Gla residues in this domain (of 45–47 residues in total) facilitate interactions with an array of calcium ions to stabilize a conformation that is required for binding to phospholipid membranes. Apart from the conantokin and vitamin K-dependent protein families, the functional role of Gla is still largely unknown. Here we report the three-dimensional structure of glacontryphan-M, in the absence and presence of calcium using two-dimensional 1H NMR spectroscopy. Calcium ion binding induces perturbations of the N-terminal residues Gla2, Ser3, and Gla4, and the Cys11-Cys5-Pro6 region of the intercysteine loop, resulting in an increased exposure and slight reorientation of the stacked aromatic rings of His8 and d-Trp7 relative to the positioning of Trp10. Considering that glacontryphan-M is a calcium-dependent antagonist for L-type voltage-gated Ca2+ channels expressed in mouse pancreatic β-cells (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar), it is possible that this structural perturbation is necessary for glacontryphan-M to interact with the Ca2+ channel in a conformation that expresses a pharmacophore similar to known L-type Ca2+ channel antagonists. The elucidation of these molecular determinants will help us to better understand the functional role of Gla within glacontryphan-M and may shed light into the potential interactions that underlie the L-type Ca2+ channel antagonism exhibited by this unique conotoxin. A complete understanding of the pharmacological activity that is attributed to the triad of hydrophobic residues, d-Trp7, His8, and Trp10, in glacontryphan-M could lead to the development of novel neuropharmacological reagents. Peptide Synthesis—Glacontryphan-M and the glutamic acid-containing analogue, glucontryphan-M, were synthesized as described by Hansson et al. (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar). The masses of the peptides were confirmed by NanoElectro Spray Ionization (NanoESI)-mass spectrometry on an API QSTAR Pulsar-I quadrapole TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada), and the peptides were sequenced by amino acid sequencing as described previously (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar). Circular Dichroism—Lyophilized glacontryphan-M was dissolved in H2O to a final concentration of 0.2 mm. Samples were adjusted to either pH 5.8 or 7.0 and treated with Chelex resin (Bio-Rad) to remove contaminant metal ions. For calcium binding studies, CaCl2 was titrated into the samples to a final concentration of 6 mm. The CD spectra were collected on a JASCO 810 spectropolarimeter (Jasco Inc., Easton, MD) equipped with a thermoelectric temperature control unit. The spectrometer was calibrated using a standard of d-(+)-10-camphor sulfonic acid. The spectra presented are the average of three wavelength scans between 230 and 340 nm using a pathlength of 1.0 cm, a dwell time of 3 s/nm and at a temperature of 25 °C. All spectra were corrected for light scatter by buffer subtraction. Results are expressed in terms of units of absorption (uA) (24Pain R. Protocols in Protein Science. John Wiley & Sons, Inc., New York1996: 7.6.1-7.6.23Google Scholar). Three-dimensional Structure Determination of Glacontryphan-M— Proton assignments were determined from two-dimensional 1H NMR data recorded over a temperature range from 5 to 25 °C on a 500 MHz Varian Unity INOVA spectrometer with a proton frequency of 499.695 MHz. The three-dimensional structures of metal-free and calcium-bound glacontryphan-M were determined from data collected at 9 °C. Samples contained 3 mm synthetic glacontryphan-M peptide in 90% H2O:10% D2O, pH 5.8 or 7.0. The carrier frequency was set to the water resonance, which was suppressed using presaturation during the preacquisition delay. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) spectra were recorded with mixing times of 300, 350, and 400 ms at 9 °C. A total of 4096 real data points were acquired in t2 and 256 time-proportional phase increments (TPPI) were implemented in t1 with a spectral width of 8000 Hz in the F2 dimension. A total of 128 summed transients were collected with a relaxation delay of 1.12 s. Additional NOESY data were acquired with relyophilized samples of glacontryphan-M that were reconstituted in 99.996% D2O (Cambridge Isotope Laboratories, Andover, MA) at a noncorrected pH of 5.8 in order to complete the assignments of resonances located near the water peak and to detect weak NOE cross-peaks. Two-dimensional TOCSY spectra were recorded with a mixing time of 45 ms at 9 °C using the MLEV-17 spin-lock sequence (25Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar). A total of 4096 real data points were acquired in t2 and 256 TPPI implemented in t1 with a spectral width of 8000 Hz in the F2 dimension. A total of 192 summed transients were collected with a relaxation delay of 1.12 s. Spectra were processed with Gaussian and sine bell functions for apodization in t2 and a shifted sine bell function in t1 using the VNMR software processing (Varian, Inc., Palo Alto, CA). All data were zero filled to 4K by 4K real matrices. A two-dimensional DQF-COSY spectrum was acquired with 4096 real data points in t2 and 640 TPPI were implemented in t1 with a spectral width of 8000 Hz in the F2 dimension. A total of 96 summed transients were collected for measurement of 3JHNα spin-spin coupling constants in order to derive dihedral torsion angles. A natural abundance 1H-13C heteronuclear single quantum coherence spectrum (HSQC) was acquired with 1024 real data points in t2 and 160 time proportional phase increments (TPPI) were implemented in t1 with a spectral width of 17591 Hz in the F1 dimension and 8000 Hz in the F2 dimension. Proton Resonance Assignments—Spin-system identifications of glacontryphan-M proton resonances in the absence of calcium and in the presence of calcium (15 molar equivalents) were completed using a combination DFQ-COSY and TOCSY experiments, which provided 1H-1H through-bond connectivities. For sequence-specific assignments, sequential dαN, dβN, and dNN NOE connectivities were obtained from NOESY experiments. The vicinal spin-spin coupling constants, 3JHNα, which were less than 6.2 Hz or greater than 8.0 Hz were used to calculate backbone ϕ torsion angles (26Clore G.M. Gronenborn A.M. Crit. Rev. Biochem. Mol. Biol. 1989; 24: 479-564Google Scholar). NOE cross-peak intensities were classified as strong, medium, or weak and converted to distance restraint upper bounds of 2.5, 3.5, and 6.0 Å, respectively (27Barsukov I.L. Lian L.-Y. Roberts G.C.K. NMR of Macromolecules: A Practical Approach. Oxford University Press, Oxford1993: 315-357Google Scholar). Scalar reference peaks were chosen from non-overlapped NH-αH NOE peaks and set to 2.8 Å distances for calibration of the entire set of distance restraints (28Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 130-199Google Scholar). Where possible rotamers of χ1 angles were categorized as either 60°, 180°, or –60° using qualitative analysis β-proton three-bond coupling (3JHαβ*) and NOE intensities, dαβ1, dαβ2, dNβ1, and dNβ2 (27Barsukov I.L. Lian L.-Y. Roberts G.C.K. NMR of Macromolecules: A Practical Approach. Oxford University Press, Oxford1993: 315-357Google Scholar). Non-stereospecifically assigned atoms were treated as pseudoatoms and assigned correction distances (28Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 130-199Google Scholar). A 1H-13C heteronuclear single quantum coherence spectrum (HSQC) was recorded to determine the prolyl peptide bond conformations using the difference in their respective Cβ and Cγ chemical shifts (29Richarz R. Wurthrich K. Biopolymers. 1978; 17: 2133-2141Google Scholar, 30Schwarzinger S. Kroon G.J.A. Foss T.R. Wright P.E. Dyson H.J. J. Biomol. NMR. 2000; 18: 43-48Google Scholar). In addition, all of the 1H-13C resonances were assigned to verify the backbone and side chain proton assignments and to resolve ambiguities in assignment of the d-Trp7, His8, and Trp10 aromatic side chain proton chemical shifts arising from chemical shift degeneracy in the proton dimension. Structure Calculations—The three-dimensional structure of metal-free glacontryphan-M was determined from a total of 339 distance restraints (142 intraresidue, 197 interresidue; 116 sequential, 51 medium range, and 30 long range) and 30 torsion angle restraints, which were entered into the distance geometry program, DGII (CVFF force field parameters) of InsightII (Accelrys, San Diego, CA) to generate a family of 50 structures using a combination of simulated annealing and distance geometry (31Havel T.F. Prog. Biophys. Mol. Biol. 1991; 56: 43-78Google Scholar). In the starting structure for molecular dynamics simulation the Cys5-Pro6 peptide bond was created in the cis conformation and the ω angle was fixed at 0° ± 10°, all other peptide bonds were created in the trans conformation. The first family of determined structures showed convergence of the orientation of the angle around the disulfide S-S bond, χ3. Greater than 80% of the convergent structures contained the right-handed disulfide bond conformation with a χ3 angle of approximately +60° (32Richardson J.S. Adv. Prot. Chem. 1981; 34: 167-339Google Scholar). These data permitted the addition of distance restraints between the cysteine α-carbons and between the β-carbon of one cysteine and the sulfur atom of the other to the restraint file (32Richardson J.S. Adv. Prot. Chem. 1981; 34: 167-339Google Scholar). The 35 conformations with the lowest restraint energy violations were used to represent the structure and the average structure for the ensemble was determined using the Analysis program of InsightII. Average root mean-squared deviation (RMSD) values following superimposition of the backbone heavy atoms of each structure with the geometric average reflected the quality of the determined family of structures. The structure determination of glacontryphan-M in the presence of 15 molar equivalents of calcium was determined in a manner similar to the methodology described above for the metal-free structure. The three-dimensional structure of calcium-bound glacontryphan-M was determined from a total of 288 distance restraints (135 intraresidue, 153 interresidue; 89 sequential, 46 medium range, and 18 long range) and 12 torsion angle restraints. Greater than 90% of the first family of determined structures showed convergence of the disulfide S-S bond orientation, χ3, therefore, we restrained the disulfide in the right-handed conformation as described above with a χ3 of approximately +60° (32Richardson J.S. Adv. Prot. Chem. 1981; 34: 167-339Google Scholar). Based on the finding that glacontryphan-M expresses a single, saturable calcium-binding site (KD 0.63 mm) (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar), the final structure calculations included six distance restraints between the Cβ, the Cγ, the Cδ1 and C1 carbons of Gla2 and Gla4, to model a bound calcium ion between Gla2 and Gla4. These distance restraints were based on the measured distances between paired Gla residues (Gla25 and Gla30) involved in the coordination of a single calcium ion in known structures of the calcium-bound human factor IX Gla domain, FIX (1–47) (33Freedman S.J. Furie B.C. Furie B. Baleja J.D. Biochemistry. 1995; 34: 12126-12137Google Scholar, 39Huang M. Furie B.C. Furie B. J. Biol. Chem. 2004; 279: 14338-14346Google Scholar). Inclusion of these restraints did not significantly perturb the protein backbone conformation or violate any of the NOE-derived distance constraints involving residues in the N terminus. The 30 conformations with the lowest restraint energy violations were used to represent the structure and the average structure for the ensemble was determined using the Analysis program of InsightII. The Cα trace and backbone heavy atoms of the average structure of calcium-bound glacontryphan-M were superimposed with the average structures of contryphan-R (7Pallaghy P.K. Melnikova A.P. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 1999; 28: 11553-11559Google Scholar), contryphan-Vn (10Massilia G.R. Eliseo T. Grolleau F. Lapied B. Barbier J. Bournaud R. Molgo J. Cicero D.O. Paci M. Schinina M.E. Ascenzi P. Polticelli F. Biochem. Biophys. Res. Commun. 2003; 303: 238-246Google Scholar), and contryphan-Sm (5Pallaghy P.K. He W. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 2000; 39: 12845-12852Google Scholar) using InsightII. To determine the high resolution structure of glacontryphan-M, a peptide having the mature glacontryphan-M sequence was synthesized with d-tryptophan at position 7, an amidated C terminus, and Gla residues at positions 2 and 4. The primary sequence and the identification of post-translational modifications of native glacontryphan-M (Table I), which was isolated and purified from the venom of C. marmoreous, were determined by automated Edman degradation and NanoESI mass spectrometry (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar). Following the synthesis and initial purification of glacontryphan-M, the Cys5-Cys11 disulfide bond was formed by oxidative refolding. The HPLC elution profiles of the natural and synthetic glacontryphan-M peptides were identical and when co-injected, the two peptides co-migrated (11Hansson K. Ma X. Eliasson L. Czerwiec E. Furie B. Furie B.C. Rorsman P. Stenflo J. J. Biol. Chem. 2003; 279: 32453-32463Google Scholar). Determination of the Structure of Glacontryphan-M using Two-dimensional NMR Spectroscopy—The solution structure of glacontryphan-M in the absence of calcium was solved by two-dimensional 1H NMR spectroscopy using NOESY, TOCSY, and DQF-COSY spectra collected at 9 °C and a pH of 5.8. Additional NOESY spectra collected at 35 °C and 15 °C and at 9 °C in fully deuterated solvent permitted the assignment of proton resonances close to the bulk solvent peak and provided additional NOE information. Proton resonances were assigned using sequential connectivities determined by correlating αH and side chain proton connectivities to backbone amide (NH) protons of neighboring residues (28Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 130-199Google Scholar). The annotated “fingerprint” region of a representative NOESY spectrum (Fig. 1) shows the sequential residue αN(i, i+1) and βN(i, i+1) connectivities that are observed throughout the peptide in the absence of calcium. In addition, medium and long-range αN(i, i+n) and βN(i, i+n) connectivities that define this well structured peptide are included. The proton resonance chemical shifts for metal-free glacontryphan-M are presented in Table II. In contrast to the published results for contryphan-Sm (5Pallaghy P.K. He W. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 2000; 39: 12845-12852Google Scholar), contryphan-R (7Pallaghy P.K. Melnikova A.P. Jimenez E.C. Olivera B.M. Norton R.S. Biochemistry. 1999; 28: 11553-11559Google Scholar), and contryphan-Vn (10Massilia G.R. Eliseo T. Grolleau F. Lapied B. Barbier J. Bournaud R. Molgo J. Cicero D.O. Paci M. Schinina M.E. Ascenzi P. Polticelli F. Biochem. Biophys. Res. Commun. 2003; 303: 238-246Google Scholar), glacontryphan-M is a single homogenous conformer exhibiting no cis to trans isomerization of the Cys-Pro peptidyl bond in aqueous solution over the temperature range investigated (9–25 °C).Table II1H chemical shift assignments for glacontryphan-M in the absence or presence of calciumResidueChemical shiftsaGiven in absence of calcium (presence of calcium in parentheses) at 9 °C, pH 5.8 in 90% H2O, 10% D2O.HNαHβHOtherppm7.455, 6.783 NH2Asn14.0202.695 (2.671)(7.440, 6.764)Gla28.980 (8.830)3.981 (4.016)2.019, 1.8112.925 (2.914) γH3.598, 3.504Scr38.046 (8.146)4.067 (4.071)(3.586, 3.500)Gla47.964 (8.164)3.992 (4.00)2.050, 1.9442.988 γHCys57.8274.2662.718, 2.343Pro63.9811.671, 0.9441.155, 0.221 γH2.953, 2.804 δHd-Trp78.351 (8.261)4.4542.898, 2.5626.807 δ1H, 9.824 ϵ1H, 7.100 ϵ3H, 7.061ζ2H, 6.693 ζ3H, 6.826 η2HHis86.459 (6.479)4.3321.073, 0.7056.725 δ2H8.382 ϵ1HPro93.8442.105, 1.8041.909, 1.835 γH3.394, 3.136 δHTrp106.0054.4033.195, 2.9256.756 δ1H, 10.043 ϵ1H 7.315 ϵ3H, 6.654 ζ2H 6.920 ζ3H, 6.709η2HCys116.807 (6.887)4.0552.800NH27.006, 7.407a Given in absence of calcium (presence of calcium in parentheses) at 9 °C, pH 5.8 in 90% H2O, 10% D2O. Open table in a new tab We initially evaluated the glacontryphan-M structure in the absence of calcium by comparing the observed proton and carbon chemical shifts for glacontryphan-M to known random coil chemical shifts for each amino acid (30Schwarzinger S. Kroon G.J.A. Foss T.R. Wright P.E. Dyson H.J. J. Biomol. NMR. 2000; 18: 43-48Google Scholar), adjusted for temperature (Fig. 2). Deviations from these random coil values for the NH proton, αH proton, and Cα carbon backbone chemical shifts provide information on the peptide backbone structure (28Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986: 130-199Google Scholar, 35Pastore A. Saudek V. J. Magn. Reson. 1990; 90: 165-176Google Scholar) being influenced considerably by neighboring residues when the peptide is structured (36Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Google Scholar). Several significant upfield (up to 2 ppm) chemical shift perturbations are observed for the NH protons of residues His8, Trp10, and Cys11 and for the αH protons of all residues within the intercys
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