Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca(2+) channels expressed in mouse pancreatic beta-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 gamma-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 (1)H 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 Cys(11)-Cys(5)-Pro(6) region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla(2) and Ser(3), moves away from the His(8) and Trp(10) aromatic rings and the alignment of the D-Trp(7) and His(8) aromatic rings with respect to the Trp(10) rings is altered. The blockage of L-type voltage-gated Ca(2+) 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 C alpha conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, 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.