Calcium and phosphorylation double-regulating caltractin initiating target protein XPC function

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Human centrin 2 (HsCen2) is an EF-hand protein crucial for centrosome duplication and separation.
• Protein phosphorylation is a key regulatory mechanism for centrin function.

Purpose of the Study:

• To investigate the structural and functional effects of HsCen2 phosphorylation by PKA.
• To elucidate the regulation of peptide p22-XPC binding to HsCen2 by phosphorylation and calcium ions.

Main Methods:

• ³¹P NMR spectroscopy to confirm phosphorylation of HsCen2 at serine 170.
• Isothermal titration calorimetry (ITC) to quantify binding affinity between HsCen2 and p22-XPC.
• Spectroscopic analysis to assess structural changes.

Main Results:

• Phosphorylation of HsCen2 by PKA significantly decreased α-helix content and exposed hydrophobic surfaces.
• Phosphorylation reduced the binding affinity of p22-XPC to HsCen2 by nearly two orders of magnitude.
• Binding between HsCen2 and p22-XPC was abolished in the absence of calcium ions and was reversible.

Conclusions:

• HsCen2 phosphorylation by PKA alters its structure and weakens its interaction with p22-XPC.
• The binding of p22-XPC to HsCen2 is regulated by both phosphorylation and calcium ions, indicating a dual control mechanism.
• These findings provide insights into the interplay between post-translational modifications (PTMs) and metal ion regulation in protein function.