Stoichiometry of the Calcineurin Regulatory Domain–Calmodulin Complex

Dunlap, Tori B.; Guo, Hou Fu; Cook, Erik C.; Holbrook, Emily; Rumi-Masante, Julie; Lester, Terrence E.; Colbert, Christopher L.; Kooi, Craig W. Vander; Creamer, T.

doi:10.1021/bi5004734

Cited by 45 publications

(66 citation statements)

References 58 publications

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“…For example, 13 C α chemical shifts (Fig. 2A) indicate a slight propensity for helix formation between residues 417–422 and 452–460, consistent with previous findings that indicate these regions adopt a helical conformation when bound to Calmodulin (Manalan and Klee 1983, Dunlap, Guo et al 2014). This propensity, however, is not borne out by 13 C β chemical shifts (Fig.…”

Section: Assignments and Data Depositionsupporting

confidence: 88%

“…For simplicity, the first three residues (385–387) and the C-terminal Histidine tag residues (469–481) are numbered as part of the sequence, even though they are not part of the wild-type RD domain. The total RD construct, containing 97 residues, corresponds to constructs used previously for the study of the RD, and purification proceeded as described previously (Dunlap, Cook et al 2013, Dunlap, Guo et al 2014). Transformed cells were incubated in 100 ml terrific broth (TB; 12 g/L tryptone, 24 g/L yeast extract, 4.0 ml glycerol, 0.17 M KH 2 PO 4 , 0.72 M K 2 HPO 4 , 75 μg/ml ampicillin and 50 μg/ml kanamycin) overnight at 37°C.…”

Section: Methods and Experimentsmentioning

confidence: 99%

“…Subsequent work has investigated CaN bound to cyclosporin A (Jin and Harrison 2002), and a solution NMR structure has been obtained for the catalytic domain of CaN bound to an NFAT-derived PVIVIT peptide (Takeuchi, Sun et al 2015). The structure of the CaM-CaN complex is of high interest (Ye, Wang et al 2008, Ye, Feng et al 2013, Dunlap, Guo et al 2014); however, no structure to date has been obtained that includes the complete CaN RD. Here, we present backbone and side-chain assignments of the RD of CaN (residues 388–468) in the solution phase, obtained using 2D and 3D NMR techniques.…”

Section: Biological Contextmentioning

confidence: 99%

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1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

et al. 2017

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Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the Ca…aN interaction, we have undertaken an NMR study of the RD of CaN. Complete 13C, 15N and 1H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of 13C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A 15N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.

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Section: Assignments and Data Depositionsupporting

confidence: 88%

Section: Methods and Experimentsmentioning

confidence: 99%

Section: Biological Contextmentioning

confidence: 99%

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1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

et al. 2017

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“…The N-domain (residue numbers 3 to 75) and C-domain (residue numbers 76 to 147) of CaN were extracted from the crystal structure (PDB ID: 4Q5U (25)). For CaN peptides, three different peptides with varying lengths and charge distributions were considered: 1) pCaN: native binding region for CaM.…”

Section: Methodsmentioning

confidence: 99%

Electrostatic control of calcineurin's intrinsically-disordered regulatory domain binding to calmodulin

Sun

Cook

Creamer

et al. 2018

Biochimica et Biophysica Acta (BBA) - General Subjects

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Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The calcineurin (CaN) regulatory domain (RD) is responsible for regulating the enzyme's phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorder-to-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating calmodulin (CaM) binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder facilitate CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.

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“…However, the molecular mechanism of CaN activation is not fully understood because the primary binding site for CaM is within an intrinsically disordered region on CaN A that has not been observed in high resolution structures of the catalytic domain of CaN A . There are crystallographic structures of (Ca 2+ ) 4 -CaM bound to a peptide representing the CaMBD sequence of the α isoform of CaN A (Ye, Wang et al 2008, Dunlap, Guo et al 2014), but no structures were reported for (Ca 2+ ) 4 -CaM bound to the CaMBD of the β isoform of the CaN A (β-CaN A ).…”

Section: Biological Contextmentioning

confidence: 99%

Backbone and side-chain resonance assignments of (Ca2+)4–calmodulin bound to beta calcineurin A CaMBD peptide

et al. 2017

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Calcineurin (CaN) is a heterodimeric and highly conserved serine/threonine phosphatase (PP2B) that plays a critical role in coupling calcium signals to physiological processes including embryonic cardiac development, NF-AT-regulated gene expression in immune responses, and apoptosis. The catalytic subunit (CaNA) has three isoforms (α, β, and γ,) in humans and seven isoforms in Paramecium. In all eukaryotes, the EF-hand protein calmodulin (CaM) regulates CaN activity in a calcium-dependent manner. The N- and C-domains of CaM (CaMN and CaMC) recognize a CaM-binding domain (CaMBD) within an intrinsically disordered region of CaNA that precedes the auto-inhibitory domain (AID) of CaNA. Here we present nearly complete 1H, 13C, and 15N resonance assignments of (Ca2+)4-CaM bound to a peptide containing the CaMBD sequence in the beta isoform of CaNA (βCaNA-CaMBDp). Its secondary structure elements predicted from the assigned chemical shifts were in good agreement with those observed in the high-resolution structures of (Ca2+)4-CaM bound to CaMBDs of multiple enzymes. Based on the reported literature, the CaMBD of the α isoform of CaNA can bind to CaM in two opposing orientations which may influence the regulatory function of CaM. Because a high resolution structure of (Ca2+)4-CaM bound to βCaNA-CaMBDp has not been reported, our studies serve as a starting point for determining the solution structure of this complex. This will demonstrate the preferred orientation of (Ca2+)4-CaM on the CaMBD as well as the orientations of CaMN and CaMC relative to each other and to the AID of βCaNA.

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Stoichiometry of the Calcineurin Regulatory Domain–Calmodulin Complex

Cited by 45 publications

References 58 publications

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

Electrostatic control of calcineurin's intrinsically-disordered regulatory domain binding to calmodulin

Backbone and side-chain resonance assignments of (Ca2+)4–calmodulin bound to beta calcineurin A CaMBD peptide

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