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Published on: December 30, 2016
Crystal structure of human 14-3-3ζ complexed with the noncanonical phosphopeptide from proapoptotic BAD
Nikolai N Sluchanko1, Kristina V Tugaeva1, Ivan Gushchin2
1A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia.
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.
Insights
The 14-3-3/BAD interaction is crucial for cell fate, but its structure is unclear. Unexpectedly, BAD binds 14-3-3 via noncanonical Ser74 phosphorylation, revealing groove plasticity.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- The proapoptotic protein BAD interacts with 14-3-3 proteins, a complex critical for regulating cell death.
- Phosphorylation of BAD controls its binding to 14-3-3, influencing apoptosis.
- The 14-3-3/BAD interaction is a potential drug target, yet lacks detailed structural characterization.
Purpose of the Study:
- To structurally characterize the interaction between 14-3-3ζ and a BAD protein fragment.
- To investigate the role of specific phosphosites, Ser74 and Ser75, in BAD binding to 14-3-3.
Main Methods:
- X-ray crystallography was employed to determine the structure of the 14-3-3ζ/BAD fragment complex at 2.36 Å resolution.
Main Results:
- The crystal structure revealed an unexpected binding mode where BAD was phosphorylated at Ser74, not the canonical Ser75 site.
- This noncanonical phosphorylation led to a positional shift and reorganization of the binding interface.
- The findings highlight the plasticity of the 14-3-3 binding groove in accommodating different peptide conformations.
Conclusions:
- The study reveals the structural basis for noncanonical binding of BAD to 14-3-3, mediated by Ser74 phosphorylation.
- This suggests functional redundancy between Ser74 and Ser75 phosphosites in regulating BAD's interaction with 14-3-3.
- The observed plasticity of the 14-3-3 groove has implications for drug design targeting this interaction.
