INVESTIGATING THE HIGH-AFFINITY AND LOW SEQUENCE SPECIFICITY OF CALMODULIN-BINDING TO ITS TARGETS

Calmodulin (CaM) is a calcium binding protein that regulates a wide ra nge of enzymes. Recently the structures of a number of complexes betwe en CaM and synthetic target peptides have been determined. The peptide s correspond to the CaM-binding domain of skeletal and smooth muscle m yosin light-chain kinase (MLCK) and calmodulin-dependent protein kinas e II alpha. Comparison of the peptide-free and peptide-bound structure s reveals that CaM undergoes a large conformational change when formin g a complex, resulting in the formation of a binding surface that prov ides for an optimal interaction with its target. In this work, the ava ilable co-ordinates of the NMR solution structure of CaM-skeletal MLCK peptide are used as a basis upon which several molecular models of bi nding are built. The detailed features of the protein's peptide bindin g surface are revealed through two-dimensional topographical projectio ns. Negatively charged margins at the binding surface extremities inte ract strongly with basic peptide residues separated by nine or ten pos itions. The binding surface core is hydrophobic and displays a groove with four deep pockets, which can accommodate bulky peptide residues a t relative positions 4 and 8 (pocket A), 11 (pocket B), 13 (pocket C), 14 and 17 (pocket D). Therefore, both electrostatic and van der Waals ' features contribute to the high affinity binding. A search for alter native peptide placements in the binding tunnel reveals the dominant r ole of specific electrostatic interactions in the binding energy. Apol ar interactions are more permissive, such that the hydrophobic side-ch ains that line the binding tunnel adapt in order to maintain favourabl e van der Waals' contacts. The model suggests that the structure can a ccommodate large peptide translations (up to 5 Angstrom) and a reverse d peptide binding mode, with a little loss in binding interaction ener gy. These calculations are compared with available experimental data, providing a structural rationale for the low sequence specificity of t he CaM target recognition.