title: Conformational changes in conantokin-G induced upon binding of calcium and magnesium as revealed by NMR structural analysis.
abstract: The apo- and metal-bound solution conformations of synthetic conantokin-G (con-G, G1Egamma gammaL5Q gamma NQgamma 10LIRgamma K15SN-CONH2, gamma = gamma-carboxyglutamic acid), an antagonist of N-methyl-D-aspartate receptor-derived neuronal ion channels, have been examined by one- and two-dimensional 1H NMR at neutral pH. A complete structure for the Mg2+-loaded peptide was defined by use of distance geometry calculations and was found to exist as an alpha-helix that spans the entire peptide. The alpha-helical nature of Mg2+/con-G was also supported by the small values (<5.5 Hz) of the 3JHNalpha coupling constants measured for amino acid residues 3-5, 8, 9, and 11-16, and the small values (<4 ppb/K) of the temperature coefficients observed for the alphaNH protons of residues 5-17. This conformation contrasted with that obtained for apo-con-G, which was nearly structureless in solution. Docking of Mg2+ into con-G was accomplished by use of the genetic algorithm/molecular dynamics simulation method, employing the NMR-derived Mg2+-loaded structure for initial coordinates in the midpoint calculations. For the 3 Mg2+/con-G model, it was found that binding of one Mg2+ ion is stabilized by oxygen atoms from three gamma-carboxylates of Gla3, Gla4, and Gla7; another Mg2+ is coordinated by two oxygen atoms, one from each of the gamma-carboxylates of Gla7; and a third metal ion through three donor oxygen atoms of gamma-carboxylates from Gla10 and Gla14. As shown from direct metal binding measurements to mutant con-G peptides, these latter two Gla residues probably stabilized the tightest binding Mg2+ ion. Circular dichroism studies of these same peptide variants demonstrated that all Gla residues contribute to the adoption of the Mg2+-dependent alpha-helical conformation in con-G. The data obtained in this investigation provide a molecular basis for the large conformational alteration observed in apo-con-G as a result of divalent cation binding and allow assessment of the roles of individual Gla residues in defining certain of the structure-function properties of con-G.
authors.affiliation: Biomolecular NMR Laboratory and the Montreal Joint Center for Structural Biology, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: T
authors.initials: T
authors.lastname: Blandl
authors.forename: M
authors.initials: M
authors.lastname: Prorok
authors.forename: S E
authors.initials: SE
authors.lastname: Warder
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: L G
authors.initials: LG
authors.lastname: Pedersen
authors.forename: F
authors.initials: F
authors.lastname: Ni
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D020916:Conotoxins; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008978:Mollusk Venoms / Q000737:chemistry / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012996:Solutions; D013329:Structure-Activity Relationship
chemical_list: D020916:Conotoxins; D008978:Mollusk Venoms; D010456:Peptides, Cyclic; D012996:Solutions; D015055:1-Carboxyglutamic Acid; C055407:conotoxin GV; D008274:Magnesium; D002118:Calcium