Zinc binding regulates amyloid-like aggregation of GAPR-1.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• The CAP superfamily, including Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins, shares a conserved CAP domain.
• A unified structure-function relationship for the CAP domain remains largely unknown.
• Several CAP proteins are known to form amyloid-like structures, particularly in lipid environments.

Purpose of the Study:

• To investigate the structural modulation of Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1) by CAP domain interactors.
• To elucidate the role of zinc ions in GAPR-1 conformational changes and subsequent amyloid-like aggregation.
• To explore if metal-ion-regulated oligomerization is a general property of CAP domain proteins.

Main Methods:

• Isothermal titration calorimetry (ITC) to assess GAPR-1's binding to zinc ions.
• Circular dichroism (CD) and tryptophan fluorescence spectroscopy to detect conformational changes.
• Trypsin digestion assays to probe structural alterations.
• Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) to analyze amyloid-like assembly formation.
• Molecular dynamics simulations to identify zinc binding sites.
• Site-directed mutagenesis of conserved histidine residues.

Main Results:

• GAPR-1 exhibits direct binding to zinc ions (Zn²⁺).
• Zn²⁺ binding induces a subtle yet significant conformational change in GAPR-1.
• This Zn²⁺-mediated conformational shift is essential for GAPR-1 oligomerization and amyloid-like structure formation in the presence of heparin.
• Molecular dynamics simulations pinpoint His⁵⁴ and His¹⁰³ as key Zn²⁺ binding sites.
• Mutating these conserved histidine residues significantly reduces amyloid-like aggregation.
• Proteins from the cysteine-rich secretory protein (CRISP) subfamily also form ThT-positive structures in vitro in a heparin- and Zn²⁺-dependent manner.

Conclusions:

• Zinc ion binding is a critical regulator of GAPR-1 structure and function, promoting amyloid-like aggregation.
• The identified Zn²⁺ binding sites (His⁵⁴, His¹⁰³) are crucial for this aggregation process.
• Metal-ion-dependent oligomerization represents a potential common structural characteristic of the CAP domain superfamily, extending to CRISP proteins.