title: Mapping of contact sites in the caldesmon-calmodulin complex.
abstract: The interaction of intact calmodulin and its four tryptic peptides with deletion mutants of caldesmon was analysed by native gel electrophoresis, fluorescence spectroscopy and zero-length cross-linking. Deletion mutants H2 (containing calmodulin-binding sites A and B) and H9 (containing sites B and B') interacted with intact calmodulin to form complexes whose stoichiometries varied from 2:1 to 1:1. The N-terminal peptides of calmodulin (TR1C, residues 1-77, and TR2E, residues 1-90) bound H2 with higher affinity than H9. At the same time H2 was less effective than H9 in binding to the C-terminal peptides of calmodulin TR2C (residues 78-148) and TR3E (residues 107-148). The N-terminal peptides of calmodulin (TR1C and TR2E) could be cross-linked to intact caldesmon and its deletion mutants H2 and H9. The similarity in the primary structures of sites A and B' of caldesmon and our measurements of the affinities of H2 and H9 to calmodulin and its peptides strongly indicate an orientation of the protein complex where sites A and B' interact with the N-terminal domain of calmodulin, whereas site B interacts with the C-terminal domain of calmodulin. The spatial organization of contact sites in the caldesmon-calmodulin complex agrees with the earlier proposed two-dimensional model of interaction of the two proteins [Huber, El-Mezgueldi, Grabarek, Slatter, Levine and Marston (1996) Biochem. J. 316, 413-420].
authors.affiliation: Department of Biochemistry, School of Biology, Moscow State University, Moscow 119899, Russia.
authors.forename: M V
authors.initials: MV
authors.lastname: Medvedeva
authors.forename: E A
authors.initials: EA
authors.lastname: Kolobova
authors.forename: P A
authors.initials: PA
authors.lastname: Huber
authors.forename: I D
authors.initials: ID
authors.lastname: Fraser
authors.forename: S B
authors.initials: SB
authors.lastname: Marston
authors.forename: N B
authors.initials: NB
authors.lastname: Gusev
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D003432:Cross-Linking Reagents; D004372:Ducks; D005895:Gizzard, Avian; D007700:Kinetics; D008961:Models, Structural; D009130:Muscle, Smooth / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins