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].
Proper evaluation of chemical cross-linking-based spatial restraints improves the precision of modeling homo-oligomeric protein complexes