Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

Christine B. Karim, L. Michel Espinoza-Fonseca, Zachary M. James, Eric A. Hanse, Jeffrey S. Gaynes, David D. Thomas, Ameeta Kelekar

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

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 β-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.

Original languageEnglish (US)
Article number14557
JournalScientific reports
Volume5
DOIs
StatePublished - Sep 28 2015

Bibliographical note

Funding Information:
The simulations also suggest that the reduced flexibility in the N-terminal region of the β-loop-β motif, combined with the favorable long-range electrostatic interaction between pSer13 and Arg30, would together result in partial masking of the BH3 domain, affecting Noxa’s ability to bind Mcl-1 and thus, its pro-apoptotic function. This prediction is clearly supported by the EPR data and by in vitro binding assays5. The addition of Mcl-1 decreased the intensity of the R (disordered) state but substantially increased the order parameter (S) of the T-state of unphosphorylated Noxa, consistent with binding. However, Mcl-1 very minimally destabilized the ordered conformation of pSer13-Noxa, supporting the lack of a binding interaction between the two proteins.

Publisher Copyright:
© 2015, Nature Publishing Group. All rights reserved.

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