Design of a phosphorylatable PDZ domain with peptide-specific affinity changes

Area of Science:

• Biochemistry
• Protein Engineering
• Synthetic Biology

Background:

• Phosphorylation is a key posttranslational modification regulating protein activity.
• Designing specific phosphorylation sites is crucial for controlling protein function.

Purpose of the Study:

• To develop a computational and experimental approach for designing novel phosphorylation sites into globular proteins.
• To engineer the Erbin PDZ domain for phosphorylation by cAMP-dependent protein kinase to modulate its function.

Main Methods:

• Combined computational design and experimental validation.
• Targeted the Erbin PDZ domain for phosphorylation by cAMP-dependent protein kinase.
• Analyzed successful and unsuccessful designs to identify design principles.

Main Results:

• Successfully designed new phosphorylation sites into the Erbin PDZ domain.
• Observed a trade-off between protein stability and phosphorylation modification.
• Demonstrated that phosphorylation can alter peptide binding affinity and specificity.
• One design introduced favorable interactions between a designed arginine and phosphoserine.

Conclusions:

• The developed strategy enables the design of phosphoswitchable protein domains.
• Engineered PDZ domains can be utilized as regulatable interaction modules in synthetic biology.
• This work opens avenues for creating diverse phosphoswitchable domains for various applications.