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14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove
C Petosa1, S C Masters, L A Bankston
1Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
Abstract:
14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.
Insights
14-3-3 proteins bind diverse signaling molecules through distinct motifs. Crystal structures reveal how these proteins accommodate both phosphorylated and unphosphorylated ligands in their binding grooves, explaining their versatile roles in signal transduction.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Signaling
Background:
- 14-3-3 proteins are crucial regulators of signal transduction, cell cycle, and apoptosis.
- They bind various ligands, including phosphorylated motifs (e.g., Raf-1, Bad) and unphosphorylated motifs (e.g., glycoprotein Ib, exoenzyme S).
Purpose of the Study:
- To elucidate the structural basis of 14-3-3 zeta isoform's interaction with both phosphorylated and unphosphorylated ligands.
- To understand the mechanism by which 14-3-3 proteins bind diverse target molecules.
Main Methods:
- X-ray crystallography was used to determine the structures of 14-3-3 zeta in complex with two distinct peptide ligands.
- Peptide ligands included a Raf-derived phosphopeptide and an unphosphorylated peptide identified through phage display.
Main Results:
- Crystal structures revealed that both phosphopeptide and unphosphorylated peptide bind to overlapping but distinct sites within a conserved amphipathic groove on 14-3-3.
- The phosphoserine of the pS-Raf-259 peptide interacted with basic residues, while the unphosphorylated R18 peptide utilized its amphipathic sequence and acidic groups to bind the same basic cluster.
- 14-3-3 proteins are dimeric, with two peptide-binding grooves oriented antiparallel.
Conclusions:
- The dual binding capability of 14-3-3's grooves allows for the accommodation of different ligand motifs.
- This structural plasticity explains how 14-3-3 proteins can mediate signal transduction by inducing homodimer or heterodimer formation in target proteins.