Metal-Thiolate Clusters in the C-Terminal Domain of Human Neuronal Growth Inhibitory Factor (GIF) ² Daniel W. Hasler, Peter Faller, and Milan Vas ˇa ´k* Biochemisches Institut der UniVersita ¨ t Zu ¨ rich, Winterthurerstrasse 190, CH-8057 Zu ¨ rich, Switzerland ReceiVed June 10, 1998; ReVised Manuscript ReceiVed August 24, 1998 ABSTRACT: Neuronal growth inhibitory factor (GIF), a metallothionein-like protein (metallothionein-3), impairs the survival and neurite formation of cultured neurons. Native GIF contains 4 Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters. However, the cluster localization is not known. In this study, the metal-thiolate clusters formed with monovalent and divalent metal ions in the C-terminal domain of human GIF [GIF(32-68)] containing 11 cysteines were investigated. The cluster formation was followed by using electronic absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy, and in the case of Cu(I) complexes also by luminescence spectroscopy at 77 K. Spectroscopic studies on the Cu(I)-GIF(32-68) complexes showed the successive formation of two air-sensitive Cu 4 S 8-9 - and Cu 6 S 11 -clusters. With Zn(II) and Cd(II) ions, a well-defined M 4 S 11 -cluster is formed in which each metal ion is tetrahedrally coordinated by cysteine thiolates. In the 113 Cd NMR spectra of 113 Cd 4 -GIF(32-68), recorded at 293 and 323 K, all four 113 Cd resonances at 672.8, 620.9, 629.6, and 564.2 ppm were observed only at 323 K. Their detection at elevated temperature indicates a conformational flexibility of this domain. Evidence for the existence of a Cd 6 -GIF(32-68) complex, contaning two more weakly bound Cd(II) ions, was also obtained. The formation of this complex requires the transformation of some originally terminal thiolates of the Cd 4 S 11 -cluster to bridging thiolates, suggesting a more accessible cluster structure. Such properties of Cd 4 -GIF(32-68) have not been observed with the Cd 4 S 11 -cluster in the isolated R-domain (amino acids 31-61) of metallothioneins. The significance of Cu- and Zn-clusters for the structure of native GIF is discussed. The neuronal growth inhibitory factor (GIF) 1 has been discovered during the studies of Alzheimer’s disease (AD). The pathology of AD brains is characterized by a progressive loss of neurons, the accumulation of extracelluar amyloid plaques, and intraneural neurofibrillary tangles (1-4). It has been hypothesized that the formation of such structures is due to the lack of neurotrophic factors (5). To test this hypothesis, the effect of AD brain extract on neonatal cortical neurons in vitro was examined by Uchida and Tomonaga (1989) (6). In these cell culture studies, the AD brain extract showed enhanced neurotrophic activity characterized by a marked dendrite sprouting and increased cell survival compared to that of normal human brain. This neuronal plasticity could be linked to the loss of a growth regulatory activity in AD brains. The protein responsible for this effect has been identified as a neuronal growth inhibitory factor (GIF). This protein is abundant in normal human brains but deficient in AD brains (7). Subsequent studies have con- firmed the growth inhibitory activity of this protein, but its deficiency in AD brains has been put in question (8). However, in recent immunohistochemical studies of the brains of patients with Down syndrome, characterized by an AD type of pathology, the loss of GIF in astrocytes around senile plaques has been demonstrated (9). GIF is a low molecular mass (7-8 kDa) cysteine- and metal-rich protein. The amino acid sequence of human GIF (68 amino acids) exhibits about 70% sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), including the preserved array of 20 cysteines (7). Molecular biological studies revealed that the GIF gene has a similar size and exon/intron organization and is clustered on the same chromosome as the MT-1 and MT-2 isoforms, leading to its classification as MT-3 (10, 11). Similar amino acid sequences to that of human GIF have also been reported for rat, mouse, porcine, equine, bovine, and canine GIF (10, 12-15). Compared to the class of mammalian MT-1/MT-2 sequences, the consensus GIF sequence shows the following prominent differences: (i) a conserved insert of a Thr occurring in the P(3)ETC(6) sequence; (ii) two conserved prolines in the C(6)-P-C-P(9) motif; and (iii) an insert of an acidic hexapeptide in the C-terminal region. Moreover, in contrast to the seven Zn usually present in MT-1/MT-2 (16), native human GIF contains four Cu and three Zn (7). Two independent studies located the N-terminal part of GIF as the biological active part of the protein (17, 18). Thus, the ² This work was supported in part by Swiss National Science Foundation Grant 32-49460.96, Stipendiendfonds der Basler Chemis- chen Industrie (D.W.H.), and EMDO Stiftung (P.F.). * To whom correspondence should be addressed at the Biochemis- ches Institut der Universita ¨t Zu ¨ rich, Winterthurerstrasse 190, CH-8057 Zu ¨rich, Switzerland. Telephone: (+41)-1-635 5552. Fax: (+41)-1- 635 6805. E-Mail: mvasak@bioc.unizh.ch. 1 Abbreviations: AD, Alzheimer’s disease; GIF, growth inhibitory factor; GIF(32-68), C-terminal part of human GIF (amino acids 32- 68); MT, metallothionein; RMT, isolated R-domain of metallothionein (amino acids 31-61); CD, circular dichroism; MCD, magnetic circular dichroism; LMCT, ligand-to-metal charge transfer; EXAFS, extended X-ray absorption fine structure. 14966 Biochemistry 1998, 37, 14966-14973 10.1021/bi9813734 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/01/1998