Paper Citing NAMD - Abstract
Karim, Christine B.; Espinoza-Fonseca, L. Michel; James, Zachary M.; Hanse, Eric A.; Gaynes, Jeffrey S.; Thomas, David D.; Kelekar, Ameeta
Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation
SCIENTIFIC REPORTS, 5 Art. No. 14557, SEP 28 2015
We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics ( MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel beta-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N- terminus and the BH3 domain. EPR showed that a spin label inserted near the N- terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.
