TOPOLOGY OF THE CALMODULIN-MELITTIN COMPLEX

The topology of the Ca2+-calmodulin-melittin ternary complex has been investigated by a combined strategy which integrates limited proteolys is and cross-linking experiments with mass spectrometric methodologies . The rationale behind the methods is that the interface regions of tw o interacting proteins are accessible to the solvent in the isolated m olecules, whereas they become protected following the formation of the complex. Therefore, when limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential Peptid e mars are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly contro lled conditions lead to the identification of spatially closed amino a cid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify c ovalently linked residues.Our results show that melittin interacts wit h calmodulin by adopting a parallel orientation, i.e. the N and C-term inal halves of the peptide are anchored to the amino and carboxy-termi nal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model o f the complex was designed and refined on the basis of the experimenta l results, supporting the above conclusions. This finding reveals a fu rther dimension to the already remarkable capability of calmodulin in binding different protein substrates, providing this protein with the capability of regulating an even larger number of enzymes. (C) 1998 Ac ademic Press Limited.