Kinase recognition by calmodulin: Modeling the interaction with the autoinhibitory region of human cardiac titin kinase

Calmodulin (CaM)-protein interactions are usually described by studying com plexes between synthetic targets of ca 25 amino acids and CaM To understand the relevance of contacts outside the protein-binding region, we investiga ted the complex between recombinant human CaM (hCaM) and P7, a 38-residue p eptide corresponding to the autoinhibitory domain of human cardiac titin ki nase (hTK). To expedite the structure determination of hCaM-P7 we relied up on the high degree of similarity with other CaM-kinase peptide complexes. B y using a combined homonuclear NMR spectroscopy and molecular modeling appr oach, we verified fur the bound hCaM similar trends in chemical shifts as w ell as conservation of NOE patterns, which taken together imply the conserv ation of CaM secondary structure. P7 was anchored to the protein with 52 ex perimental intermolecular contacts. The hCaM-P7 structure is very similar t o known CaM complexes, but the presence of NOE contacts outside the binding cavity arrears to he novel. Comparison with the hTK crystal structure indi cates that the P7 charged residues all correspond to accessible side-chains , while the putative anchoring hydrophobic side-chains are partially buried . To test this finding, we also modeled the early steps of the complex form ation between Ca2+-loaded hCaM and hTK. The calculated trajectories strongl y suggest the existence of an "electrostatic funnel", driving the long-rang e recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residu es remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca2+/hCaM, favoring the intrasteric r egulation of the kinase. Furthermore; our structure, the first of CaM bound to a peptide derived From a kinase whose three-dimensional structure is kn own, suggests that special care is needed in the choice of template peptide s to model protein-protein interactions. (C) 2001 Academic Press.