The complex structure of calmodulin bound to a calcineurin peptide

Abstract

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.

Crystallographic basis for calcium regulation of sodium channels

“…2C), suggesting the C lobe blocks access of the N lobe, as shown in other CaM complexes (18). The structure and ITC data therefore highlight the C lobe as the major and likely sole anchor point to the DIII-IV linker, and suggest that Ca 2+ /CaM may bridge different segments with the N lobe binding elsewhere, reminiscent of Ca 2+ /CaM bound to calcineurin (19) or Ca 2+ -activated K + channels (20). To date, a number of long Q-T type 3 (LQT3) cardiac disease mutations have been identified in the DIII-IV linker, and the positions of five of these can be directly observed in the crystal structure (Fig.…”

A Calcineurin Docking Motif (LXVP) in Dynamin-related Protein 1 Contributes to Mitochondrial Fragmentation and Ischemic Neuronal Injury

“…CaN is composed of two subunits, a catalytic calcineurin A subunit (CNA) and a calcium-sensitive regulatory calcineurin B subunit (CNB) (20). Upon binding of Ca 2ϩ to the EF hands of CNB and Ca 2ϩ /CaM to CNA, CNA undergoes a conformational change that dislodges its autoinhibitory domain from the catalytic cleft (21)(22)(23). This conformational change also exposes a composite hydrophobic docking surface made of the interface between CNA and CNB, which mediates binding to substrates contain-* This work was supported, in whole or in part, by National Institutes of Health ing an LXVP motif (24,25).…”

Calmodulin in Complex with Proteins and Small Molecule Ligands: Operating with the Element of Surprise; Implications for Structure-Based Drug Design

“…In a 2:2 complex, CaM displays a native-like extended conformation while the calcineurin binding sequence is in alpha-helical conformation. However, the N-terminal lobe from one CaM and the C-terminal lobe from another form a combined binding site to trap the peptide, in a unique Xshaped arrangement [43,44]. The binding sites thus created are quite similar to that of the most commonly seen bent complexes.…”