Molecular basis of calmodulin-dependent calcineurin activation

Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation by Ca2+ is in part driven by binding of calmodulin (CaM) to a Calmodulin (CaM)-recognition motif within the phosphatases regulatory domain (RD); how-ever, secondary interactions between CaM and the Calcineurin (CaN) regulatory domain may be necessary to fully activate CaN [1]. Specifically, it has been shown that the CaN regulatory domain folds upon CaM binding and that there is a region C-terminal to the canonical CaM-binding region, the distal helix, that assumes an helix fold and contributes to activation [1]. We hypothesized in Dunlap et al [1] that this putative helical distal helix is capable of binding CaM in a region distinct from the canonical CaM binding region (CaMBR) site, whereby CaN is activated. To test this hypothesis, we utilized molecular simulations including replica-exchange molecular dynamics, protein-protein docking and computational mutagene-sis to model distal helix conformations. From these simulations we have isolated a potential binding site on CaM (site D) that facilitates moderate affinity inter-protein interactions that may attenuate CaN auto-inhibition. Further, molecular simulations of the distal helix A454E mutation demonstrated weakened distal helix/CaM interactions that were previously shown to impair CaN activity. K30E and G40D mutations of CaM at site D presented similar de-creases in binding affinity predicted by simulations. The prediction was correlated with a phosphatase assay in which these two mutants show reduced CaN activity. This study there-fore provides a potential structural basis for the role of secondary CaM/CaN interactions in mediating CaN activation.