Structure-function studies of a lipid-binding class I MHC-like protein may lead to a possible treatment for type 2 diabetes

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Strategic Research Priority: Bioscience for HealthAbstract The WHO predicts that diabetes will be the seventh leading cause of death in 2030. Zinc a2 glycoprotein (ZAG) is an adipokine that breaks down human fat cells (lipolysis). There is growing evidence from biochemical studies to suggest that ZAG is relevant to diabetes, however its biochemical mechanism involving lipid binding to its MHC-like groove remains unknown. This project will use state-of-the-art structural, biophysical and computational methods based on lipid-binding studies to ZAG to identify the molecular mechanism of ZAG-induced lipolysis. The student will benefit from unique cross-disciplinary training opportunities in a well-equipped immunology laboratory. Project Background: ZAG possesses a class I MHC-like protein fold with an open apical groove between its a1 and a2 domain helices. However ZAG is distinct from MHC by being soluble and not anchored to plasma membranes, and associates with prolactin-inducible protein rather than B2-microglobulin. The original crystal structure of human ZAG revealed unidentifiable electron density in its major groove (PDB 1T7Z). We found that this density is polyethylene glycol, a crystallization adjuvant (Ref.4). We also showed that ZAG contains one tightly-bound zinc ion [Ref.1], predicted to lie close to the a1 and a2 domain helices. This year, by combining fluorescent titrations and fluorescent-detected analytical ultracentrifugation, we have unexpectedly shown that there are at least two distinct lipid binding sites in the ZAG groove (Ref.2). Aim: The lipid-binding properties of ZAG will be identified to uncover its molecular mechanism of action. Plan of investigation: We will use E. coli recombinant and human plasma-purified ZAG. By fluorescent-tagging recombinant ZAG, we can identify the ZAG-containing fraction from plasma, and isolate ZAG from this. From this, we can create an antibody or lipid-coated affinity column to isolate native ZAG from human plasma. First we will use LC-MS mass spectrometry to identify ZAG's intrinsic lipid using plasma-purified ZAG, adding a plasma lipid-rich/protein-poor fraction separated by centrifugation to this. Chemical-lipid libraries will screen for tightly-bound lipids. Second, we will re-crystallise ZAG, and soak the crystals in a lipid-rich plasma fraction in order to facilitate the binding of the lipid with the strongest affinity. The crystal structure will identify the bound lipid and its conformation. Crystals will also be exposed to zinc to identify the strong zinc binding site in ZAG. Third, having identified the tightest bound lipids, competition experiments with fluorescent-labelled lipids will identify the affinities of the bound lipids observed to bind to the ZAG groove.