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 proteolysis and cross-linking experiments with mass spectrometric methodologies. The rationale behind the methods is that the interface regions of two interacting proteins are accessible to the solvent in the isolated molecules, 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 peptide maps are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly controlled conditions lead to the identification of spatially closed amino acid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify covalently linked residues. Our results show that melittin interacts with calmodulin by adopting a parallel orientation, i.e. the N and C-terminal halves of the peptide are anchored to the amino and carboxy-terminal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model of the complex was designed and refined on the basis of the experimental results, supporting the above conclusions. This finding reveals a further 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.
Scaloni
A
A
Centro Internazionale di Servizi di Spettrometria di Massa, CNR-Università di Napoli, Napoli, "Federico II", 80131, Italy.
Miraglia
N
N
Orrù
S
S
Amodeo
P
P
Motta
A
A
Marino
G
G
Pucci
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008555:Melitten / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin
D002147:Calmodulin; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008555:Melitten; D014357:Trypsin