Zn-alpha(2)-glycoprotein (ZAG) is a soluble
lipid-mobilizing factor associated with
cancer cachexia and is a novel
adipokine. Its X-ray crystal structure reveals a poly(
ethylene glycol) molecule, presumably substituting for a higher affinity natural
ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the
peptide binding groove in
class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic
ligands that may relate to the
protein's signaling function and that the natural
ligands are probably (
polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic
ligands. We also identify
amino acid positions that are involved in
ligand binding and those that control the shape and exposure to
solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged
amino acid adjacent to the
ligand. Replacing this
amino acid with
alanine abolished
ligand binding and closed the groove to
solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic
ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like
proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.