Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms.

Area of Science:

• Protein-protein interactions
• Structural biology
• Human papillomavirus (HPV) oncoproteins

Background:

• 14-3-3 proteins are abundant, highly conserved human proteins that regulate numerous cellular processes by binding phosphorylated motifs.
• Human papillomaviruses, particularly HPV, utilize oncoproteins like E6 to subvert host cell functions, often involving interactions with host proteins.

Purpose of the Study:

• To investigate the structural basis and druggability of 14-3-3 isoform binding to HPV E6 oncoproteins.
• To understand the affinity hierarchy of 14-3-3 isoforms when binding to E6 and other phosphoproteins.

Main Methods:

• X-ray crystallography to determine protein structures.
• Fluorescence polarization assays to measure binding affinities.
• Site-directed mutagenesis to probe protein interactions.
• Fusicoccin-mediated modulation to study protein complex dynamics.

Main Results:

• 14-3-3 isoforms exhibit differential binding affinities to variant and mutated phospho-motifs of HPV E6 oncoproteins and the unrelated protein RSK1.
• A conserved relative affinity ranking (K_D ratios) was observed across different 14-3-3/phosphoprotein interactions, including E6 and RSK1.
• This affinity hierarchy is consistent with known 14-3-3 interactions across a wide range of targets, as evidenced by literature and complexome data.

Conclusions:

• The conserved binding hierarchy of 14-3-3 isoforms provides a framework for predicting their engagement with phosphoproteins in various human tissues.
• Cellular concentrations of 14-3-3 proteins may be collectively regulated to manage phosphorylation signaling, explaining their varied expression in tissues and tumors.