The Metal-free and Calcium-bound Structures of a γ-Carboxyglutamic Acid-containing Contryphan from Conus marmoreus, Glacontryphan-M*

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.