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.
|Number of pages||16|
|State||Published - Oct 1 2019|
Bibliographical noteFunding Information:
Research reported in this publication was supported by the Maximizing Investigators’ Research Award (MIRA) (R35) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Grant R35GM124977. This work was also supported by Grant R01HL138579.
This work used the Extreme Science and Engineering Discovery Environment (XSEDE), (91) which is supported by National Science Foundation under Grant ACI-1548562.
© 2019 American Chemical Society.
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