pubs.acs.org/Biochemistry Published on Web 05/08/2009 r 2009 American Chemical Society 5700 Biochemistry 2009, 48, 5700–5707 DOI: 10.1021/bi900366p Effects of Zn 2+ , Ca 2+ , and Mg 2+ on the Structure of Zn 7 Metallothionein-3: Evidence for an Additional Zinc Binding Site † Gabriele Meloni, ‡ Thomas Polanski, ‡ Oliver Braun, and Milan Va  s ak* Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland ‡ These authors contributed equally to this work Received March 4, 2009; Revised Manuscript Received May 6, 2009 ABSTRACT: Human metallothionein-3 (Zn 7 MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain, where it plays an important role in the homeostasis of the essential metal ions Cu + and Zn 2+ . Like other mammalian metallothioneins (MT-1 and -2), the protein contains a M II 3 (CysS) 9 and a M II 4 (CysS) 11 cluster localized in two independent protein domains linked by a flexible hinge region. However, there is a substantially increased number of acidic residues in MT-3 (11 residues) compared with MT-2 (four residues) which may act as binding ligands for additional metal ions. In this study, the binding of Zn 2+ , Ca 2+ , and Mg 2+ to human Zn 7 MT-3 and its mutant lacking an acidic hexapeptide insert, Zn 7 MT- 3 Δ55-60 , was investigated and compared with the binding of Zn 7 MT-2. By using spectroscopic and spectro- metric techniques, we demonstrate that one additional Zn 2+ binds with an apparent binding constant (K app ) of ∼100 μM to Zn 7 MT-3 and Zn 7 MT-3 Δ55-60 , but not to Zn 7 MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn 8 MT-3 is immediate and is accompanied by a decrease in the Stokes radius (R s ). The changes in the R s suggest a mutual approach of both protein domains. The fast binding of Zn 2+ is followed by a slow time-dependent protein dimerization. The binding of Zn 2+ to Zn 7 MT-3 is specific as in the presence of Ca 2+ and Mg 2+ only an alteration of the R s of Zn 7 MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is discussed. The brain has the highest content of zinc of all organs with an average total zinc concentration estimated to be approximately 150 μM(1). This trace element plays structural, catalytic, or regulatory roles in numerous enzymes and other proteins (2). Apart from its importance in protein complexes, the zinc ion is closely involved in intracellular signaling and neurotransmission. In the mammalian brain, 10-15% of the total Zn 2+ is localized in presynaptic vesicles of zinc-enriched neurons (ZEN), 1 a subclass of glutamatergic neurons (3). ZEN are present in many regions of the central nervous system (CNS) and are especially abundant in the hippocampus. The best established function for released Zn 2+ into the synaptic cleft is the modulation of the glutamate and GABA receptors on postsynaptic cells (4). Cellular zinc uptake is controlled by a family of membranous zinc transporter proteins called ZIPs, whereas the zinc transporters ZnTs, which belong to the cation diffusion facilitator family (CDF), mediate zinc efflux (5). While in the synaptic vesicles of ZEN an approximately millimolar concentration of chelatable Zn 2+ was found, that in the cytosol is in the subnanomolar range. Low intracellular free Zn 2+ concentrations in neurons are maintained by the action of cytosolic metal binding proteins, the most abundant of which is metallothionein-3 (MT-3). This metalloprotein reversibly binds seven Zn 2+ ions with high affinity through an array of 20 Cys residues. Zn 7 MT-3, also termed the neuronal growth inhibitory factor (GIF), occurs intra- and extracellularly and shows neuroinhibitory activity in vitro that distinguishes it from the widely expressed MT-1 and MT-2 isoforms (6, 7). Thus, MT-3, but not MT-1 or -2, antagonizes the ability of Alzheimer’s disease (AD) brain extract to stimulate survival and neuritic sprouting of cultured neurons (6, 8). This extracellular bioactivity led to the hypothesis that Zn 7 MT-3 may be involved in pathogenic processes leading to AD. The observa- tion that Zn 7 MT-3 protects the neuronal cells from the toxic effect of amyloid-β (Aβ), by abolishing the production of reactive oxygen species (ROS) and related cellular toxicity caused by the redox cycling of Aβ-Cu(II), strongly supports its protective role in AD (9, 10). † This work was supported by Swiss National Science Foundation Grant 3100A0-111884 to M.V. *To whom correspondence should be addressed. Telephone: +41-44- 635-55-52. Fax: +41-44-635-59-05. E-mail: mvasak@bioc.uzh.ch. 1 Abbreviations: ZEN, zinc-enriched neurons; CNS, central nervous system; GABA, γ-aminobutyric acid; ZIP, Zrt- and Irt-like proteins; ZnT, zinc transporters; CDF, cation diffusion facilitator; MT, metal- lothionein; GIF, growth inhibitory factor; AD, Alzheimer’s disease; Aβ, amyloid-β; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; ESI-MS, electrospray ionization mass spectrometry; DTP, 2,2 0 -dithio- pyridine; EDTA, ethylenediaminetetraacetic acid; CD, circular dichro- ism; Tris, tris(hydroxymethyl)aminomethane; SEC, size exclusion chromatography; NMR, nuclear magnetic resonance; LMCT, ligand- to-metal charge transfer.