The complex structure of calmodulin bound to a calcineurin peptide.

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.