Strong and weak zinc binding sites in human zinc-α2-glycoprotein

• Zinc-α2-glycoprotein (ZAG) is an adipokine.
• ZAG has one strong zinc binding site and up to 15 weak zinc binding sites.
• The strong zinc binding site is predicted to lie close to the α1 and α2 helical groove.
• Weak zinc binding to holo-ZAG causes precipitation which is reversed by EDTA.
• Zinc binding to the fluorescent fatty acid probe DAUDA is enhanced by holo-ZAG.

Zinc-α2-glycoprotein (ZAG) is an adipokine with an MHC class I-like protein fold. Even though zinc causes ZAG to precipitate from plasma during protein purification, no zinc binding has been identified to date. Using mass spectrometry, we demonstrated that ZAG contains one strongly bound zinc ion, predicted to lie close to the α1 and α2 helical groove. UV, CD and fluorescence spectroscopies detected weak zinc binding to holo-ZAG, which can bind up to 15 zinc ions. Zinc binding to 11-(dansylamino) undecanoic acid was enhanced by holo-ZAG. Zinc binding may be important for ZAG binding to fatty acids and the β-adrenergic receptor.