Calmodulin-Calcineurin Interaction beyond the Calmodulin-Binding Region Contributes to Calcineurin Activation.
Biochemistry
; 58(39): 4070-4085, 2019 10 01.
Article
em En
| MEDLINE
| ID: mdl-31483613
ABSTRACT
Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation is in part driven by the binding of calmodulin (CaM) to a CaM recognition region (CaMBR) within CaN's regulatory domain (RD). However, secondary interactions between CaM and the CaN RD may be necessary to fully activate CaN. Specifically, it is established that the CaN RD folds upon CaM binding and a region C-terminal to CaMBR, the "distal helix", assumes an α-helix fold and contributes to activation [Dunlap, T. B., et al. (2013) Biochemistry 52, 8643-8651]. We hypothesized in that previous study that this distal helix can bind CaM in a region distinct from the canonical CaMBR. To test this hypothesis, we utilized molecular simulations, including replica-exchange molecular dynamics, protein-protein docking, and computational mutagenesis, to determine potential distal helix-binding sites on CaM's surface. We isolated a potential binding site on CaM (site D) that facilitates moderate-affinity interprotein interactions and predicted that mutation of site D residues K30 and G40 on CaM would weaken CaN distal helix binding. We experimentally confirmed that two variants (K30E and G40D) indicate weaker binding of a phosphate substrate p-nitrophenyl phosphate to the CaN catalytic site by a phosphatase assay. This weakened substrate affinity is consistent with competitive binding of the CaN autoinhibition domain to the catalytic site, which we suggest is due to the weakened distal helix-CaM interactions. This study therefore suggests a novel mechanism for CaM regulation of CaN that may extend to other CaM targets.
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1
Base de dados:
MEDLINE
Assunto principal:
Calmodulina
/
Calcineurina
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article