The Recognition of Calmodulin to the Target Sequence of Calcineurin-A Novel Binding Mode

Calcineurin (CaN) is a Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase, which plays essential roles in many cellular and developmental processes. CaN comprises two subunits, a catalytic subunit (CaN-A, 60 kDa) and a regulatory subunit (CaN-B, 19 kDa). CaN-A tightly binds to CaN-B in the presence of minimal levels of Ca²⁺, but the enzyme is inactive until activated by CaM. Upon binding to CaM, CaN then undergoes a conformational rearrangement, the auto inhibitory domain is displaced and thus allows for full activity. In order to elucidate the regulatory role of CaM in the activation processes of CaN, we used NMR spectroscopy to determine the structure of the complex of CaM and the target peptide of CaN (CaNp). The CaM/CaNp complex shows a compact ellipsoidal shape with 8 α-helices of CaM wrapping around the CaNp helix. The RMSD of backbone and heavy atoms of twenty lowest energy structures of CaM/CaNp complex are 0.66 and 1.14 Å, respectively. The structure of CaM/CaNp complex can be classified as a novel binding mode family 1-18 with major anchor residues Ile³⁹⁶ and Leu⁴¹³ to allocate the largest space between two domains of CaM. The relative orientation of CaNp to CaM is similar to the CaMKK peptide in the 1-16 binding mode with N- and C-terminal hydrophobic anchors of target sequence engulfed in the hydrophobic pockets of the N- and C-domain of CaM, respectively. In the light of the structural model of CaM/CaNp complex reported here, we provide new insight in the activation processes of CaN by CaM. We propose that the hydrophobic interactions between the Ca²⁺-saturated C-domain and C-terminal half of the target sequence provide driving forces for the initial recognition. Subsequent folding in the target sequence and structural readjustments in CaM enhance the formation of the complex and affinity to calcium. The electrostatic repulsion between CaM/CaNp complex and AID may result in the displacement of AID from active site for full activity.