Crystal structure of human 14-3-3ζ complexed with the noncanonical phosphopeptide from proapoptotic BAD

Nikolai N Sluchanko; Kristina V Tugaeva; Ivan Gushchin; Alina Remeeva; Kirill Kovalev; Richard B Cooley

doi:10.1016/j.bbrc.2021.10.053

Crystal structure of human 14-3-3ζ complexed with the noncanonical phosphopeptide from proapoptotic BAD

Biochem Biophys Res Commun. 2021 Oct 26:583:100-105. doi: 10.1016/j.bbrc.2021.10.053. Online ahead of print.

Authors

Nikolai N Sluchanko¹, Kristina V Tugaeva², Ivan Gushchin³, Alina Remeeva³, Kirill Kovalev⁴, Richard B Cooley⁵

Affiliations

¹ A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia. Electronic address: nikolai.sluchanko@mail.ru.
² A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia.
³ Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701, Dolgoprudny, Russia.
⁴ Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701, Dolgoprudny, Russia; European Molecular Biology Laboratory, 22607, Hamburg, Germany.
⁵ Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA.

PMID: 34735870
DOI: 10.1016/j.bbrc.2021.10.053

Abstract

Several signaling pathways control phosphorylation of the proapoptotic protein BAD and its phosphorylation-dependent association with 14-3-3 proteins in the cytoplasm. The stability of the 14-3-3/BAD complex determines the cell fate: unphosphorylated BAD escapes from 14-3-3, migrates to the mitochondria and initiates apoptosis. While the 14-3-3/BAD interaction represents a promising drug target, it lacks structural characterization. Among several phosphosites identified in vivo, Ser75 and Ser99 of human BAD match the consensus sequence RXXpSXP recognized by 14-3-3 and, therefore, represent canonical 14-3-3-binding sites. Yet, BAD contains other serines phosphorylatable in vivo, whose role is less understood. Here, we report a 2.36 Å crystal structure of 14-3-3ζ complexed with a BAD fragment which includes residues Ser74 and Ser75, both being substrates for protein kinases. While the BAD peptide is anchored to 14-3-3 by phosphoserine as expected, the BAD peptide was unexpectedly phosphorylated at Ser74 instead of Ser75, revealing noncanonical binding within the amphipathic groove and leading to a one-step positional shift and reorganization of the interface. This observation exemplifies plasticity of the amphipathic 14-3-3 groove in accommodating various peptides and suggests the redundancy of Ser74 and Ser75 phosphosites with respect to binding of BAD to 14-3-3.

Keywords: Apoptosis regulation; Crystallography; Phosphorylation; Protein chimera; Protein-peptide complex.