Structure of Calmodulin Bound to a Calcineurin Peptide:  A New Way of Making an Old Binding Mode^,

Abstract

Calcineurin is a calmodulin-binding protein in brain and the only serine/threonine protein phosphatase under the control of Ca2+/calmodulin (CaM), which plays a critical role in coupling Ca2+ signals to cellular responses. CaM up-regulates the phosphatase activity of calcineurin by binding to the CaM-binding domain (CBD) of calcineurin subunit A. Here, we report crystal structural studies of CaM bound to a CBD peptide. The chimeric protein containing CaM and the CBD peptide forms an intimate homodimer, in which CaM displays a native-like extended conformation and the CBD peptide shows alpha-helical structure. Unexpectedly, the N-terminal lobe from one CaM and the C-terminal lobe from the second molecule form a combined binding site to trap the peptide. Thus, the dimer provides two binding sites, each of which is reminiscent of the fully collapsed conformation of CaM commonly observed in complex with, for example, the myosin light chain kinase (MLCK) peptide. The interaction between the peptide and CaM is highly specific and similar to MLCK.

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…The model was further extended to include calmodulin binding to its targets. It was shown that autoinhibitor domains of CaMKII and PP2B bound to the same, open, conformation of calmodulin (22,23). Therefore, we introduced the binding of both CaMKII and PP2B to calmodulin in the R state, independent of the number of bound calcium ions.…”

Structural Studies of Soybean Calmodulin Isoform 4 Bound to the Calmodulin-binding Domain of Tobacco Mitogen-activated Protein Kinase Phosphatase-1 Provide Insights into a Sequential Target Binding Mode

“…In the course of our work we also noted that these fusion proteins seem to prevent aggregation, and that they provided more stable NMR samples than the regular noncovalent Ca 2ϩ -CaM CaMBD-peptide complexes. Previously, Jia and coworkers (45) have determined a crystal structure for Ca 2ϩ -CaM fused with the CaMBD of calcineurin via a poly-Gly linker and they uncovered a unique complex, in which CaM forms a ''head to tail'' dimer through the two bound CaMBDs. More recently, the same group has demonstrated that the crystal structures of their CaM fusion protein and that of the complex with a noncovalently bound CaMBD peptide were essentially identical (C␣ r.m.s.d.…”

TRAF3 negatively regulates calcineurin‐NFAT pathway by targeting calcineurin B subunit for degradation

“…pET-21a empty vector (EV) was bought from Novagen (Madison, WI). pET21a-CaM was constructed by our own faculty (18). pcDNA3-HA-TRAF3 was generous gift from Micheal Karin (University of California San Diego, La Jolla, CA) (19).…”

“…pcDNA3-HA-TRAF3 was used as the template and the following primers were used to obtain slice variant as indicated: for pcDNA3-TRAF3-C (T3C, aa 314-543) construction forward primer, 5 0 -ACTGAATTCCCCTTCCGGCAGAACTG-3 0 ; reverse primer, 5 0 -CTCGAGTCAGGGATCGGGCAGATCCGA-3 0 ; for pcDNA3-TRAF3-N (T3N, aa 1-313) construction forward primer 5 0 -GTGGTGGAATTCATGGAGTCGA-3 0 ; reverse primer 5 0 -GAC TCGAGTCACCGGATCTCC-3 0 . Previously, we have reported a two-round PCR procedure for construction of CaM fusion protein (18). The same PCR strategy was adopted for construction of pET21a-CaM-TRAF3-C, which contains a continuous DNA sequence coding for CaM, a thrombin cleavage site, the TRAF domain of TRAF3 (TRAF3 amino acids: 314-543) and a 63His tag.…”