A single amino acid replacement results in the Ca2+-induced self-assembly of a helical conantokin-based peptide.
Conantokins are short (17-27 amino acid residues), gamma-carboxyglutamate (Gla)-rich peptide components of the venoms of marine snails of the genus Conus. They display high apo and/or Ca(2+)-induced helicity and act as potent and selective inhibitors of the N-methyl-d-aspartate receptor (NMDAR). We have previously established that one of the conantokins, conantokin-G (con-G), self-associates in the presence of Ca(2+) with high specificity for antiparallel chain orientation [Dai, Q., Prorok, M., and Castellino, F. J. (2004) J. Mol. Biol. 336, 731-744]. The dimerization appears to be driven by interhelical Ca(2+) coordination between the following residue pairings: Gla(3)-Gla(14)('), Gla(7)-Gla(10)('), Gla(10)-Gla(7)('), and Gla(14)-Gla(3)('). A second member of the conantokin family, conantokin-T (con-T), shares sequence identity with con-G at 8 of 21 amino acids, including 4 Gla residues. These similarities notwithstanding, several primary and secondary structural differences exist between con-T and con-G. Particularly notable is that con-T contains a Lys, rather than a Gla, at position 7. Moreover, unlike con-G, con-T does not undergo Ca(2+)-triggered self-assembly. In the present study, sedimentation equilibrium ultracentrifugation is employed to demonstrate that a single amino acid replacement analogue of con-T, con-T[K7gamma], assumes a dimeric superstructure in the presence of Ca(2+) at pH values consistent with the ionization of Gla carboxylate groups. Furthermore, HPLC-monitored thiol-disulfide folding and rearrangement assays with Cys-containing con-T variants suggest that the relative chain alignment preference in the noncovalent complex is antiparallel. Our results suggest that interchain Ca(2+) coordination in con-T[K7gamma] is incumbent upon an "i, i + 4, i +7, i +11" arrangement of Gla residues, as occurs in native con-G.
Department of Chemistry and Biochemistry and the W. M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Qiuyun
Q
Dai
Francis J
FJ
Castellino
Mary
M
Prorok
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary
D020916:Conotoxins; D036341:Intercellular Signaling Peptides and Proteins; D008978:Mollusk Venoms; D010455:Peptides; C063571:conantokin-T; D015055:1-Carboxyglutamic Acid; C055407:conotoxin GV; D008239:Lysine; D002118:Calcium