Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

Human zinc-α2-glycoprotein (ZAG) is a 42 kDa adipokine which regulates body fat mass and is associated with cachexia and obesity. ZAG belongs to the major histocompatibility complex class I protein family and binds long-chain polyunsaturated fatty acids in its groove formed from the α1 and α2 domains. To identify the molecular basis of its lipid-binding function, we determined the first crystal structure at 2.49 Å resolution for fatty acid-bound ZAG, where the ligand was the fluorescent 11-(dansylamino)undecanoic acid (DAUDA). The 192 kDa crystallographic asymmetric unit contained six ZAG and eight fatty acid molecules in unique conformations. Six fatty acid molecules were localised to the ZAG grooves, where their tails were bound in two distinct conformations. The carboxylate groups of three fatty acids projected out of the groove, while the fourth was hydrogen bonded with R73 inside the groove. Other ligand-residue contacts were primarily hydrophobic. A new fatty acid site was revealed for two further DAUDA molecules at the ZAG α3 domains. Following conformational changes from unbound ZAG, the α3 domains formed tetrameric β-barrel structures lined by fatty acid molecules that doubled the binding capacity of ZAG. Analytical ultracentrifugation revealed that ZAG in solution was a monomer in the absence of DAUDA, but formed small amounts of tetramers with DAUDA. By showing that ZAG binds fatty acids in different locations, we demonstrate an augmented mechanism for fatty acid binding in ZAG that is distinct from other known fatty acid binding proteins, and may be relevant to cachexia.