Dax Fu Biology Department, 463 Brookhaven National Laboratory Upton, NY 11973-5000 tel: (631) 344-4208 lab: (631) 344-4952 fax: (631) 344-3407 dax@bnl.gov The Group: Dax Fu (631) 344-4208 Jin Chai (631) 344-3419 Xiongying Tu (631) 344-3550

Research Summary The challenge:  Recent advances in our understanding of zinc biology have placed this once obscure metal in the center stage, rivaling the biological importance of calcium. Zinc transporters utilize the transmembrane electrochemical potential to move zinc ions across the membrane barrier. Our biochemical studies have shown that the binding affinity of a zinc transporter is much lower than expected for effective Zn(II) equilibrium binding in vivo, while the timescale of Zn(II) transport is millisecond, vastly faster than most metalloproteins that typically take hours or even days to release bound Zn(II). It is not clear how this remarkable thermodynamic-kinetic capacity is built into zinc transporter structures, which support Zn(II) acquisition against steep thermodynamic gradients, rapid Zn(II) mobility across membranes, and yet extraordinary Zn(II)-selectivity over similar divalent cations several orders of magnitude more abundant in vivo. The goal:  We seek to understand chemical principles governing selective binding and energized movement of zinc ions in membrane transporters. Our fundamental research will facilitate drug discovery efforts targeting human zinc transporters. The human zinc transporter-8 (ZnT-8 or SLC308A) is exclusively expressed in insulin-producing beta-cells, where its activity is required for insulin packing and secretion. ZnT-8 is a major risk factor for type-2 diabetes, an obesity-related disease that is expected to affect 30% of the American population born in 2000. The druggability of ZnT-8 has been established recently. We have filed a patent application on homology modeling of ZnT-8 and its virtual docking of potential drugs. The approaches:  We have used stopped-flow fluorometry to show how Zn(II) binding to a metal transporter triggers transmembrane movements of bound Zn(II) (8). Binding competition analysis has confirmed that the active binding site is indeed highly selective for Zn(II) over similar divalent cations (5). The Zn(II) binding reaction has been studied directly by titration calorimetry (7), and coordination residues have been identified by mutation-function analysis (3). The transporter structure has been characterized by light-scattering photometry (6), and determined in atomic detail by x-ray crystallography (1). Zinc binding sites revealed in the crystal structure provide insights into the mechanism of Zn(II) binding and transport. Another complementary ongoing study is to understand the structural basis of substrate selectivity in the membrane channel of aquaporins. We have determined the crystal structure of a glycerol-conducting channel GlpF (9), and a homologous water-conducting channel AqpZ (4). The structural mechanism of water/glycerol selectivity has been reviewed recently (2).

The structures of GlpF and YiiP have been solved by MIR/AS/AD phasing at 2.2 and 3.8 Å resolution respectively, while AqpZ was solved by molecular replacement at 3.2 Å resolution.

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