Substrate recognition principles for the PP2A-B55 protein phosphatase.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The protein phosphatase 2A catalytic subunit with the B55 regulatory subunit (PP2A-B55) is crucial for eukaryotic signaling pathways.
• Understanding how PP2A-B55 selects its diverse substrates remains a significant challenge in molecular biology.

Approach:

• Integrated AlphaFold modeling with high-resolution mutational scanning to elucidate substrate binding mechanisms.
• Employed deep learning for protein design to create a specific peptide inhibitor of PP2A-B55-substrate interactions.
• Utilized the designed inhibitor to probe PP2A-B55 function in cellular processes.

Key Points:

• PP2A-B55 substrates bind via conserved alpha-helical motifs, sharing key amino acid determinants for hydrophobic and electrostatic interactions.
• A novel peptide inhibitor was designed using deep learning, demonstrating high specificity and potency against PP2A-B55 substrate binding.
• The inhibitor enabled the discovery that PP2A-B55 regulates the nuclear exosome targeting complex via interaction with RBM7's alpha-helical module.

Conclusions:

• Established an evolutionary conserved mechanism for PP2A-B55 substrate recognition involving alpha-helices.
• Developed a powerful tool (peptide inhibitor) for dissecting PP2A-B55 functions.
• Uncovered a novel regulatory role of PP2A-B55 in the nuclear exosome targeting pathway, impacting RNA processing and degradation.