The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin

Grigoriu, Simina; Bond, Rachel; Cossio, Pilar; Chen, Jennifer A.; Ly, Nina; Hummer, Gerhard; Page, Rebecca; Cyert, Martha S.; Peti, Wolfgang

doi:10.1371/journal.pbio.1001492

Cited by 128 publications

(210 citation statements)

References 56 publications

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“…Recently, Grigoriu et al [24] reported the first high-resolution structure of CN α bound to a physiological binding partner, the protein inhibitor A238L from African swine fever virus. The structure of the CN-A238L complex reveals that the interface between these two proteins consists of two discontinuous contact regions.…”

Section: The Cna-cnb Interface Is a General Recognition Site For Aismentioning

confidence: 99%

“…Although the structure of CN bound to a bona fide substrate has yet to be determined, the structure of the CN-A238L complex provides the structural basis for the computational model of CN with a phosphosubstrate bound to its active site [24]. The pRII peptide (a 19-amino-acid phosphopeptide) contains a typical LxVP motif that is required for its recognition and efficient dephosphorylation by CN.…”

Section: The Cna-cnb Interface Is a General Recognition Site For Aismentioning

confidence: 99%

“…The pRII peptide (a 19-amino-acid phosphopeptide) contains a typical LxVP motif that is required for its recognition and efficient dephosphorylation by CN. Grigoriu et al [24] used bias-exchange metadynamics MD to generate the first computational model of CN in complex with the RII phosphopeptide. In this model, LDVP of pRII interacts with the LxVP-binding pocket and phospho-Ser95 interacts with the CN active site.…”

Section: The Cna-cnb Interface Is a General Recognition Site For Aismentioning

confidence: 99%

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Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Wang

et al. 2016

Cell Res

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The Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CN), a heterodimer composed of a catalytic subunit A and an essential regulatory subunit B, plays critical functions in various cellular processes such as cardiac hypertrophy and T cell activation. It is the target of the most widely used immunosuppressants for transplantation, tacrolimus (FK506) and cyclosporin A. However, the structure of a large part of the CNA regulatory region remains to be determined, and there has been considerable debate concerning the regulation of CN activity. Here, we report the crystal structure of full-length CN (β isoform), which revealed a novel autoinhibitory segment (AIS) in addition to the well-known autoinhibitory domain (AID). The AIS nestles in a hydrophobic intersubunit groove, which overlaps the recognition site for substrates and immunosuppressant-immunophilin complexes. Indeed, disruption of this AIS interaction results in partial stimulation of CN activity. More importantly, our biochemical studies demonstrate that calmodulin does not remove AID from the active site, but only regulates the orientation of AID with respect to the catalytic core, causing incomplete activation of CN. Our findings challenge the current model for CN activation, and provide a better understanding of molecular mechanisms of CN activity regulation.

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Section: The Cna-cnb Interface Is a General Recognition Site For Aismentioning

confidence: 99%

Section: The Cna-cnb Interface Is a General Recognition Site For Aismentioning

confidence: 99%

Section: The Cna-cnb Interface Is a General Recognition Site For Aismentioning

confidence: 99%

See 1 more Smart Citation

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Wang

et al. 2016

Cell Res

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“…By contrast, LxVP peptides bind to a hydrophobic groove formed by the CN A and B subunits that is accessible only after Ca 2+ /calmodulin binding activates the enzyme and releases an autoinhibitory peptide (AIS) from this site 10 . Engagement of the LxVP binding site is required for dephosphorylation and is thought to orient the phosphorylated residue toward the catalytic center 11 . In combination with an immunophilin, FK506 and CysA binds to this LxVP-binding site, thus inhibiting CN by blocking its interaction with NFAT and other substrates 8,11,12 .…”

Section: Introductionmentioning

confidence: 99%

“…Engagement of the LxVP binding site is required for dephosphorylation and is thought to orient the phosphorylated residue toward the catalytic center 11 . In combination with an immunophilin, FK506 and CysA binds to this LxVP-binding site, thus inhibiting CN by blocking its interaction with NFAT and other substrates 8,11,12 . Unfortunately, inhibition of the ubiquitous CN in non-immune tissues often leads to kidney failure, diabetes, pain, or seizures 13 , presumably due to interaction with as-yet-unidentified substrates.…”

Section: Introductionmentioning

confidence: 99%

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

Nguyen

Roy

Harink

et al. 2018

Preprint

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Weak, transient binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drive cellular signaling. Mapping the energy landscape of such interactions is essential for deciphering signaling networks and developing therapeutic inhibitors, but is complicated by technical challenges associated with quantitatively measuring weak interactions in high-throughput. Here, we synthesize peptide libraries on spectrally encoded beads with each peptide sequence uniquely linked to a spectral code (MRBLE-pep), allowing high-throughput quantification of protein-peptide binding via imaging. Using computational modeling and MRBLEpep assays, we map the affinity landscape for human calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, interacting with a known SLiM (PxIxIT). We find that PxIxIT recognition depends critically on flanking residues and is regulated by post-translational modifications. Using this information, we designed PxIxIT peptides with unprecedented affinity and therapeutic potential that strongly inhibit CN function in vivo.

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Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus

Juvvadi

Pemble

et al. 2016

FEBS Letters

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Calcineurin heterodimer, comprised of the catalytic (CnaA) and regulatory (CnaB) subunits, localizes at the hyphal tips and septa to direct growth, septation, and disease in the human pathogen Aspergillus fumigatus. Here we discovered a novel motif (FMDVF) required for this critical CnaA septal localization, including residues Phe368, Asp370 and Phe372 overlapping the cyclosporine A-cyclophilin A binding domain, CnaB-binding helix and the FK506-FKBP12 binding pocket. Mutations in adjacent residues Asn367, Trp374 and Ser375 confer FK506 resistance without impacting CnaA septal localization. Modeling A. fumigatus CnaA confirmed that the FMDVF motif forms a bridge between the two known substrate binding motifs, PxIxIT and LxVP, and concurrent mutations (F368A D370A; F368A F372A) in the FMDVF motif disrupt CnaA-substrate interaction at the septum.

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The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin

Abstract: Structural analyses show that a viral protein and immunosuppressant drugs inhibit the phosphatase calcineurin by preventing substrate binding, and provide a model of a phosphatase engaged with its substrate.

Cited by 128 publications

References 56 publications

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus

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