title: Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation.
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.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors.forename: Christine B
authors.initials: CB
authors.lastname: Karim
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors.forename: L Michel
authors.initials: LM
authors.lastname: Espinoza-Fonseca
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: James
authors.affiliation: Department of Laboratory Medicine and Pathology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Hanse
authors.affiliation: College of Biological Sciences, University of Minnesota, Minneapolis, MN 55455.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Gaynes
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Department of Laboratory Medicine and Pathology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN, 55455, USA
authors.forename: Ameeta
authors.initials: A
authors.lastname: Kelekar
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000737:chemistry / Q000378:metabolism; D010455:Peptides; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D064549:Myeloid Cell Leukemia Sequence 1 Protein; C498702:PMAIP1 protein, human; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins