title: The roles of individual gamma-carboxyglutamate residues in the solution structure and cation-dependent properties of conantokin-T.
abstract: The solution structure of the Ca2+-loaded conantokin-T (con-T), a gamma-carboxyglutamate (Gla)-containing 21-residue peptide (NH2-G1EgammagammaY5QKMLgamma10NLRgammaA15EVKKN20A-CONH2,gam ma = Gla), has been elucidated by use of distance geometry calculations with experimental distances derived from two-dimensional 1H NMR spectroscopy. An end-to-end alpha-helix was the dominant conformation in solution, similar to that of apo-con-T, except that reorientation of several side chains occurred in the Ca2+-coordinated complex. The most notable examples of this were those of Gla10 and Gla14, which were more optimally positioned for complexation with Ca2+. In addition to the stabilization offered to the alpha-helix by Ca2+ binding, hydrophobic clustering of the side chains of Tyr5, Met8, Leu9, and Leu12, and ionic interactions between Lys7 and Gla3/Gla10 and between Arg13 and Gla14, along with hydrogen bonding between Gln6 and Gla10, were among the side chain interactions likely playing a significant role in maintenance of the alpha-helical conformation. Docking of Ca2+ in the con-T structure was accomplished using genetic algorithm-molecular dynamics simulation approaches. The results showed that one Ca2+ ion is most likely coordinated by four side chain oxygen atoms, two each from Gla10 and Gla14. Another bound Ca2+ ion has as its donor sites three oxygen atoms, two from Gla3 and one from Gln6. To examine the functional roles of the individual Gla residues, a series of variant peptides have been synthesized with Ala substituted for each Gla residue, and several properties of the resulting variants have been examined. The data obtained demonstrated the importance of Gla10 and Gla14 in stabilizing binding of the highest affinity Ca2+ site and in governing the conformational change induced by Ca2+. The critical nature of Gla3 and Gla4 in inhibition of the spermine-induced potentiation of the binding of MK-801 to open ion channels of the N-methyl-D-aspartate receptor was established, as well as the role of Gla4 in stabilizing the apo-con-T alpha-helical conformation.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: S E
authors.initials: SE
authors.lastname: Warder
authors.forename: M
authors.initials: M
authors.lastname: Prorok
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
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 / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D020916:Conotoxins; D016291:Dizocilpine Maleate / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D017207:Rats, Sprague-Dawley; D012996:Solutions; D013329:Structure-Activity Relationship
chemical_list: D020916:Conotoxins; D018691:Excitatory Amino Acid Antagonists; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D008670:Metals; D008978:Mollusk Venoms; D010455:Peptides; D012996:Solutions; C063571:conantokin-T; D015055:1-Carboxyglutamic Acid; D016291:Dizocilpine Maleate; D002118:Calcium