Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Peptide conformational flexibility is crucial for peptide-macromolecular interactions.
• Understanding the flexibility of protein kinase inhibitor alpha (PKI alpha) is essential for comprehending its interaction with cAMP-dependent protein kinase (cAPK).

Purpose of the Study:

• To assess the alpha-carbonyl backbone flexibility of PKI alpha in solution and when bound to the catalytic subunit of cAPK.
• To determine the extent of the cAPK-binding domain within the PKI alpha peptide.

Main Methods:

• Time-resolved fluorescence anisotropy was used to measure the conformational flexibility of three single-site PKI alpha mutants (V3C, S28C, S59C) labeled with fluorescein iodoacetamide (FI).
• Analysis of anisotropy decay profiles using a model-free nonassociative biexponential equation.
• Comparison of flexibility measurements for free PKI alpha mutants versus those bound to the catalytic subunit of cAPK.

Main Results:

• Free PKI alpha mutants exhibited similar anisotropy decays, indicating high disorder and flexibility due to local backbone movements.
• Binding to the cAPK catalytic subunit reduced whole-body tumbling for all mutants.
• The flexibility of FI-V3C- and FI-S28C-PKI alpha was significantly reduced upon binding, while FI-S59C-PKI alpha remained largely flexible, suggesting the binding domain extends to at least Ser28 but not beyond.

Conclusions:

• The cAPK-binding domain of PKI alpha extends from its N-terminus to residues beyond Ser28.
• The segment around Ser59 is not part of the cAPK-binding domain and remains flexible even when PKI alpha is bound to cAPK.
• Conformational flexibility plays a significant role in the interaction between PKI alpha and cAPK.