Conformational and functional variability supported by the BPTI fold: Solution structure of the Ca2+ channel blocker calcicludine

Calcicludine, a 60-amino acid protein isolated from the green mamba venom, has been recently identified as blocking a large set (i.e., L-, N- and P-ty pe) of Ca2+ channels, The three-dimensional structure of calcicludine has b een det-ermined by NMR and molecular modeling using a data set of 723 unamb iguous and 265 ambiguous distance restraints, as 33 Phi, and 13 chi(1) dihe dral angle restraints. Analysis of the 15 final structures (backbone root-m ean-square deviation = 0.6 Angstrom) shows that calcicludine adopts the Kun itz-type protease inhibitor fold. Its three-dimensional structure is simila r to that of snake K+ channel blockers dendro-toxins. Conformational differ ences with protease inhibitors and dendrotoxins are localized in the 3(10) helix and loop 1 (segments 1-7 and 10-19), the extremity of the beta-hairpi n (segment 27-30), and loop 2 (segment 39-44). These regions correspond to the functional sites of bovine pancreatic trypsin inhibitor (BPTI) and dend rotoxins, The positioning of the N-terminal segment 1-7 relative to the res t of the protein is characteristic of calcicludine. The involvement of this segment and the positively charged K31 at the tip of the beta-hairpin in t he biological activity of calcicludine is discussed. Proteins 1999;34:520-5 32. (C) 1999 Wiley-Liss, Inc.