MINIREVIEW Neuronal growth-inhibitory factor (metallothionein-3): structure–function relationships Zhi-Chun Ding*, Feng-Yun Ni  and Zhong-Xian Huang Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai, China Introduction Metallothioneins (MTs), first discovered in horse kidney in 1957 by Margoshes & Vallee, are a family of small ( 7 kDa), cysteine-rich and metal-binding proteins [1]. In mammals, four subfamily MTs (i.e. MT1, MT2, MT3 and MT4), have been identified [2]. MT1 and MT2 are ubiquitous isoforms found in most organs and play criti- cal roles in essential metal homeostasis and heavy metal ions detoxification [3]. By contrast, MT3 and MT4 are specifically expressed in the central nervous system and the stratified squamous epithelia, respectively [2]. MT3, first isolated and identified as a neuronal growth-inhibitory factor (GIF), has a distinct biologi- cal activity of inhibiting the out-growth of rat embry- onic cortical neurons in the presence of Alzheimer’s disease (AD) brain extracts, a function not shared by MT1 or MT2 [4]. As a member of the MT family, GIF exhibits approximately 70% sequence similarity with those well-studied mammalian MTs, including: a pre- served array of 20 cysteine residues; and two domains, each of which wrap around a metal–thiolate cluster Keywords metallothionein (MT); mutation; neuronal growth-inhibitory factor (GIF); structure– function relationship Correspondence Z X. Huang, Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai 200433, China Fax: 86 21 65641740 Tel.: 86 21 65643973 E-mail: zxhuang@fudan.edu.cn Present address *Immunology ⁄ Immunotherapy Program & Cancer Center, Medical College of Georgia, Augusta, GA 30909, USA  Department of Bioengineering, Rice University, Houston, TX 77005, USA (Received 5 January 2010, revised 5 March 2010, accepted 18 March 2010) doi:10.1111/j.1742-4658.2010.07716.x Neuronal growth-inhibitory factor (GIF), also named metallothionein-3, inhibits the outgrowth of neuronal cells. Recent studies on the structure of human GIF, carried out using NMR and molecular dynamics simulation techniques, have been summarized. By studying a series of protein-engi- neered mutants of GIF, we showed that the bioactivity of GIF is modu- lated by multiple factors, including the unique TCPCP motif-induced characteristic conformation, the solvent accessibility and dynamics of the metal–thiolate cluster, and the domain–domain interactions. Abbreviations AD, Alzheimer’s disease; DTNB, 5,5¢-dithiobis-(1-nitrobenzoic acid); GIF, neuronal growth-inhibitory factor; hGIF, human neuronal growth-inhibitory factor; MT, metallothionein; NO, nitric oxide; pDB, protein data bank; rlMT2, rat liver MT2; sGIF, sheep neuronal growth-inhibitory factor; SNOC, S-nitrosocysteine. 2912 FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS (a three-metal cluster, M(II) 3 S 9 , in the N-terminal b-domain, and a four-metal cluster, M(II) 4 S 11 , in the C-terminal a-domain). However, there are two inserts in GIF that are not present in MT1 or MT2: a threo- nine at position 5 and a glutamate-rich hexapeptide near the C-terminus. Additionally, all known GIF sequences contain a conserved CPCP(6–9) motif, which is absent in all other members of the MT family (Fig. 1). The neuronal growth-inhibitory activity is quite unique in the mammalian MT family. We have long pondered how nature could utilize such a simple pro- tein (of only 68 amino acids) to fulfill such compli- cated functions in the central nervous system. However, the exact molecular basis of the bioactivity of GIF remains elusive. It has been reported that the neuronal growth-inhibitory activity of GIF is mainly associated with its b-domain, and the single a-domain does not show any growth-inhibitory activity [5,6]. As a new protein the structure is mostly concerned. Consequently, much effort has been devoted towards determining the structure of GIF. During the past 15 years or so, the crystal structure of rat liver MT2 (rlMT2) has been the only crystal structure obtained of MT binding to divalent metals [7], and it serves as the starting point for our laboratory to study the structure of human GIF (hGIF); to date there are no crystallographic data on GIF, possibly because GIF is so dynamic that it is difficult to crystallize. NMR was broadly applied to structural studies of MTs, and there are almost 20 entries in the protein data bank (PDB) on the structure of MTs (the majority of which are structures of MT1 ⁄ MT2) binding to divalent metal ions. The metal-to-cysteine connectivities were mostly verified by 2D 1 H- 113 Cd heteronuclear multiple quan- tum coherence (HMQC) experiments [8,9], illustrating that the connectivities were the same as those in the crystal structure of rlMT2 [7]. One point worthy of mention is that each domain of MT was refined indi- vidually because no or insufficient interdomain NOE signals were obtained to address the problem of the interaction between two domains. The progress of structural studies on GIF has mainly been made using NMR and molecular dynamics simulation. Structure of the a-domain of GIF The structures of the a-domain of rat GIF and hGIF were solved by Armitage [10] and our group [9], from NOE cross-peaks, using NMR spectroscopy [9]. Most interestingly, the hexapeptide insertion EAAEAE(55– 60), located near the C-terminus, was modeled using a group of possible conformations because of the lack of NOE signals in this region (Fig. 2). Spatially, this insertion was far from the metal–thiolate cluster, implying that it was less restricted and therefore had a A B Insertion of EAAEAE(55–60)? Insertion of EAAEAE(55–60)? Insertion of Thr5 ? Insertion of Thr5 ? CPCP(6–9)? CPCP(6–9)? Fig. 1. (A) Amino acid sequence alignments of human MT1a, MT1g, MT2a, MT4 and some mammalian GIFs. Twenty conserved cysteine residues are highlighted. The distinctive sequence differences of hGIF from other MT isoforms include an insertion of Thr5, a conservative CPCP(6–9) sequence and insertion of the charged hexapeptide EAAEAE(55–60). (B) Crystal structure of rlMT2 (PDB entry: 4MT2). The sequence dissimilarities of hGIF from other MT isoforms are located in the rlMT2 structure and labeled with a question mark. The zinc ions are shown as grey spheres, the cadmium ions are shown as green spheres and the sulfur atoms are shown as yellow spheres; this color scheme is also used in the other figures. Z C. Ding et al. Structure–reactivity–function study of GIF FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS 2913 tendency to adopt alternative conformations. The dis- order in the insertion region of hGIF was considered to make the whole protein more unstable than MT1 or MT2. [11] Structure and dynamics of the b-domain of hGIF Based on the proton-detected 2D 1 H- 15 N heteronuclear single quantum coherence (HSQC) spectroscopy, the 15 N backbone amide-relaxation parameters were deter- mined for 18 residues in the b-domain of hGIF [9]. These relaxation parameters showed that only the N-terminal 12 residues were more flexible than other regions of the protein, implying that the TCPCP(5–9) sequence at the N-terminus might contribute to the dynamics of the b-domain of hGIF. However, the structure of the b-domain of hGIF remained unsolved by NMR spectroscopy because insufficient amounts of medium-range and long-range NOE signals were available. The structure of the b-domain of hGIF was predicted using molecular dynamics simulation [12]. It was found that the peptides near the N-terminus (residues 1–13) in hGIF folded differently from those in rlMT2; in particular, a characteristic conformation of the TCPCP(5–9) sequence was formed in the b- domain of hGIF, where both Pro7 and Pro9 faced out- wards with their five-member rings arranged almost in parallel, while Thr5 was at the opposite side of the two rings. The specific folding of the TCPCP(5–9) sequence, together with the constraints from the metal–thiolate cluster, made the peptides at the two ends of the TCPCP(5–9) sequence twisted (Fig. 3A1,B1). This characteristic conformation around the TCPCP(5–9) sequence in hGIF was suggested to provide an interacting interface for protein–protein interactions [12–14]. The other structural feature found in the predicted structure of the b-domain of hGIF was the hydrogen- bond network located around the first five N-terminal residues and the fragment from residues 23 to 26, which was different from that found in the simulated structure of the b-domain of rlMT2 [12]. In rlMT2 there were two hydrogen bonds, one between Asp2 and Lys25 and one between Asn4 and Gln23 making the whole structure compact (Fig. 3A2). However, the insertion of Thr5 into hGIF interrupted these two interactions, and Thr5 formed a hydrogen bond with Asp2, pushing Lys26 (equivalent to Lys25 in rlMT2) away from Asp2. Meanwhile, Lys26 in hGIF formed hydrogen bonds with Glu4 and Gly24 (equivalent to Gln23 in rlMT2) (Fig. 3B2). These local structural arrangements that occurred in hGIF resulted in a loose conformation between the fragment near the N-termi- nus and the fragment from residues 23 to 26, therefore inducing the more exposed state of the metal–thiolate cluster. This structural feature illustrates how the inser- tion of Thr5 induces the formation of the distinct hydrogen-bond network in hGIF compared with that in rlMT2, and provides a structural basis for the sig- nificance of Thr5 in hGIF [15]. Interdomain interaction of hGIF Based on the backbone 1 H chemical shifts between the a-domain of the holo-hGIF and the single a-domain, some residues (including Ser36, Pro39, Ala40, Glu41 and Ala46) were identified to be involved in the inter- domain interaction [16]. However, no further conclu- sions could be made on the interdomain interaction by NMR unless structure of the b-domain of hGIF could be identified. It was found that in the predicted struc- ture of the holo-hGIF, all these residues mentioned above were located around the linker region and faced towards the b-domain (Fig. 4A) [12,16], implying that EAAEAE(55–60) EAAEAE(55–60) Gln68 Gln68 Lys32 Lys32 Fig. 2. Solution structure of the a-domain of hGIF (PDB entry: 2F5H). A group of minimized structures are superimposed to show clearly that the EAAEAE(55–60) insertion is structurally disordered and extending outwards. The cadmium ions are shown as green spheres and the sulfur atoms are shown as yellow spheres. Structure–reactivity–function study of GIF Z C. Ding et al. 2914 FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS the results from simulation studies are consistent with those from NMR studies. The simulated structure of hGIF and its mutant [the D55-60 hGIF mutant produced by the deletion of EA- AEAE(55–60)] also disclosed the interdomain interac- tion mode at the atomic level, which helped to elucidate the structural basis of the relevance of the hexapeptide insertion to the biological function of hGIF [16]. The first point was that the interdomain interaction modes were not exactly the same in hGIF and rlMT2 (Fig. 4A,B). The common features were that Lys32 in hGIF (equivalent to Lys31 in rlMT2) faced into the interior of the b-domain to neutralize the negative charge of the B-cluster, thus stabilizing the b-domain; and Ser33 in hGIF (equivalent to Ser32 in rlMT2) formed a hydrogen bond with Cys38 (equiv- alent to Cys37 in rlMT2) to make the a-domain more stable. The differences lay in the fact that the addi- tional hydrogen bond between Lys31 and Glu41 in hGIF made the fragment around residue 41 closer to the linker region, while Lys30 in rlMT2 (equivalent to Lys31 in hGIF) had no direct interaction with Gly40 (equivalent to Glu41 in hGIF). The second point was that the EAAEAE(55–60) sequence of hGIF would affect the interaction between the linker region and the a-domain of hGIF. As shown in the simulated structure of the D55-60 mutant of hGIF, the two hydrogen bonds found in the wild-type hGIF (between Lys31 and Glu41 and between Ser33 and Cys38) fell apart (Fig. 4C) and ultimately this would have a critical influence on the structure of the b-domain of hGIF through the change of the hydrogen- bond network. In the wide-type hGIF, the hydrogen bond between Lys32 and Cys22 would enable the move- ment of the fragment around Cys22 towards the linker region, therefore resulting in a more open conformation between the N-terminal residues and the fragment from residues 23 to 26, which would make the B-cluster more exposed to solution. While in the D55-60 mutant of hGIF, the hydrogen bond between Lys32 and Cys22 broke and the distance between them increased, instead, Lys32 formed a hydrogen bond with Cys20, implying A1 B1 A2 B2 Fig. 3. (A1) and (A2) Simulated structure of the b-domain of rlMT2. (B1) and (B2) Pre- dicted structure of the b-domain of hGIF. Panels A1 and A2 are rotated in the same view to show the twisted conformation of the first 13 residues from the N-terminus in hGIF compared with the smooth conforma- tion in rlMT2. Panels B1 and B2 are pre- sented in the same view to show a looser conformation between the fragment near the N-terminus and the fragment from resi- dues 23 to 26 in hGIF compared with that in rlMT2. The cadmium ions are shown as green spheres and the sulfur atoms are shown as yellow spheres. The red regions stress the differences. Z C. Ding et al. Structure–reactivity–function study of GIF FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS 2915 that Lys32 in this mutant would not induce the structural re-arrangement in the b-domain that was observed in the wild-type hGIF (Fig. 4D1,D2). These results interpret structurally how the hexapeptide EAAEAE(55–60) in hGIF exerts its function by affect- ing the conformation of the b-domain. Based upon the molecular structure of GIF, we have conducted a systematic mutational study on the struc- ture–property–reactivity–function relationship of GIF. These results will provide us with valuable data to understand the molecular mechanism of the neuronal growth-inhibitory activity of GIF. The role of the conserved TCPCP motif The CPCP motif was the first segment demonstrated to be indispensible for the neuronal growth-inhibitory activity of GIF [6,17]. The bioactivity of GIF is com- pletely abolished by a double mutation from Cys6- Pro7-Cys8-Pro9 to either Cys6-Ser7-Cys8-Ala9 (the P7S ⁄ P9A mutant) found in MT1 and MT2 or Cys6- Thr7-Cys8-Thr9 (the P7T ⁄ P9T mutant) [6,17]. 113 Cd NMR data showed that both the wide-type GIF and the P7S ⁄ P9A mutant exhibit four major and three minor resonances between 590 and 680 ppm at 298 K, corresponding to the four Cd 2+ ions in the a-domain and the three Cd 2+ ions in the b-domain, respectively. However, upon a temperature increase to 323 K, the three minor resonances of the b-domain of the P7S ⁄ P9A mutant partially recovered, and such a tem- perature-induced effect was not observed in wide-type GIF [17]. Hence, Hasler et al. proposed that mutating the CPCP motif of GIF to CSCA alters the dynamics of the b-domain and eliminates the bioactivity of GIF [17]. As mentioned previously, the two prolines induce constraints on the CPCP motif and make it form a characteristic conformation, where the two proline resi- dues are at the same side of the protein, both facing outwards, and the two five-member rings of prolines are arranged almost in parallel [12]. Such a conforma- tion was proposed to function as ‘the characteristic conformation’, providing an interaction surface for protein–protein interactions [13,14], which is thought to be one possible mechanism for the bioactivity of GIF. In order to test whether the CPCP motif is suffi- cient for the bioactivity of GIF, Vasak and cowork- ers introduced the CPCP motif into the neuronal inactive mouse MT1 (the S6P ⁄ S8P mutant), and examined its inhibitory activity [18]. Quite unexpect- edly, the S6P ⁄ S8P mutant of mouse MT1 did not show any inhibitory activity. However, introduction of a unique Thr5 insert before the S6P ⁄ S8P motif in the modified mouse MT1 restored the neuronal bio- activity [18]. The neuronal assay results undoubtedly reflect that the CPCP motif alone is insufficient for the bioactivity of GIF, and that both the Thr5 insert and the CPCP motif are necessary for the neuronal bioactivity of GIF. The 113 Cd NMR results showed that the acquisition of GIF bioactivity in the A B C D1 D2 Fig. 4. Simulated structure of rlMT2 (A), hGIF (B), the D55-60 mutant of hGIF (C), the b-domain of hGIF (D1) and the b-domain of the D55-60 mutant of hGIF (D2). The EAAEAE(55–60) insertion in hGIF is shown in red. Comparison between panels A and B clearly shows that the interdomain interaction modes are different in rlMT2 and hGIF. Comparison between panels B and C shows that the deletion of EAAEAE(55–60) in hGIF would change the interdomain interaction modes in hGIF. Comparison between panels D1 and D2 shows that the deletion of EAAEAE(55–60) in hGIF would affect the hydrogen-bond network in the b-domain of hGIF. The cadmium ions are shown as green spheres and the sulfur atoms are shown as yellow spheres. The red regions stress the differences. Structure–reactivity–function study of GIF Z C. Ding et al. 2916 FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS mutants of mouse MT1 is paralleled by an increase in conformational flexibility and dynamics in the N-terminal b-domain [18]. Apparently, this result agrees well with their previous conclusion that the structure ⁄ cluster dynamics is pivotal to the bioactivity of GIF [17]. Although Romero-Isart et al. pointed out the impor- tance of the Thr5 insert, the precise role played by Thr5 insertion in the bioactivity of GIF was not clear. To address this, Cai et al. constructed three mutants: the T5S and T5A mutants, with the aim to examine the role of hydroxyl group; and the DT5 mutant [15]. Bioassay results showed that the T5A and DT5 mutants have almost no inhibitory activity, which agrees well with previous reports [18]. By contrast, the T5S mutant shows an inhibitory activity comparable to that of the wild-type hGIF, indicating the impor- tance of the hydroxyl group Xaa5 [15]. The simulation results showed that the hydroxyl group of Thr5 induces formation of the distinct hydrogen-bond net- work in the b-domain of hGIF compared with that in MT2, making the structure of the b-domain of hGIF much looser than that of MT2 [12], which agrees well with the experimental data [15]. Thus, an extra residue inserted neighboring the CPCP(6–9) motif should be necessary to regulate the conformation of the protein backbone to the biologically active state [15]. Notably, Chung et al. found that sheep GIF (sGIF), which has an ACPCP(5–9) motif instead of the TCPCP(5–9) motif present in hGIF, still retains about 60% of bioactivity [19]. However, a previous study clearly showed that the bioactivity of GIF is almost completely abolished by replacing Thr5 with Ala5 [15,18]. This contradiction raises the question of why the bioactivity of sGIF is unaffected by replacement of Thr5 with Ala5. After comparison of the amino acid sequence of sGIF with that of hGIF, it was found that sGIF appears to contain three fewer cysteine residues, owing to deletion of the sequence Ser-Cys-Cys (nor- mally found at positions 33-35 of hGIF) and the replacement of a cysteine residue (normally found at position 30) with serine. However, both the Cd 2+ titra- tion and ESI-MS results showed that sGIF binds seven metal ions with the overall metal-to-thiolate ratio of Cd 7 S 17 . These seven metal ions were wrapped into two separate metal–thiolate clusters by the polypeptide chain of sGIF: one M 3 cluster and one M 4 cluster, in which the M 3 cluster was less stable than the M 4 clus- ter. Unexpectedly, it was found that non-sulfur ligands might participate in the coordination of metal ions. The composition of this novel cluster is substantially differ- ent from other, hitherto unknown, mammalian MTs. Moreover, spectroscopic and biochemical studies showed that the whole structure and dynamic proper- ties of sGIF, as well as the solvent accessibility and sta- bility of the metal–thiolate clusters, were quite different from those of hGIF. Taken together, we proposed that in sGIF the critical role of the hydroxyl group might be partly compensated by its ‘unusual structure’ and dynamic properties of the protein [unpublished data]. The effect of domain–domain interactions It was reported that the neuronal growth-inhibitory activity of hGIF arises from its b-domain, especially the TCPCP-induced characteristic conformation and dynamic properties, while the a-domain is not directly involved in neuronal growth-inhibitory activity [5,6,17,18]. However, it has been well documented that the two domains of MT do not work independently, and domain–domain interactions do exist and affect the properties and reactivity of each domain [20–22]. Furthermore, our results, and those of other bioassays, showed that the inhibitory activity of the single b-domain is less effective than that of intact hGIF on a molar basis [6,23]. Hence, it is suggested that the a-domain might play some important roles in the neu- ronal growth-inhibitory activity of hGIF. To confirm this assumption, Ding et al. constructed two domain- hybrid mutants, in which the a-domain of hGIF was replaced with either the b-domain of hGIF [the b(MT3)-b(MT3) mutant] or the a-domain of hMT1g [the b(MT3)-a(MT1) mutant] [23]. It was found that the metal-binding ability and solvent accessibility of the Cd 3 S 9 cluster of the b-domain of the b(MT3)-b(MT3) mutant decreased significantly compared with those of hGIF, while the b(MT3)-a(MT1) mutant showed biochemical properties similar to those of hGIF [23]. Interestingly, bioassay data showed that the b(MT3)- b(MT3) mutant exhibited reduced activity, while the b(MT3)-a(MT1) mutant had similar activity, confirm- ing that the a-domain is not dispensable for the neuro- nal growth-inhibitory activity of hGIF [23]. Therefore, we suggest that although the single a-domain does not exhibit any neuronal growth-inhibitory activity, it does play an important role in modulating the stability of the metal–thiolate cluster and conformation of the b-domain by domain–domain interactions, thus altering zinc homeostasis in the brain and influencing the bioactivity [23]. The role of the EAAEAE insert The main sequence differences between GIF and MT1 ⁄ MT2 are the TCPCP motif in the b-domain and Z C. Ding et al. Structure–reactivity–function study of GIF FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS 2917 the EAAEAE insert in the a-domain (Fig. 1). Many investigations have been employed on the structural and biological function of the unique TCPCP motif. By contrast, the biochemical properties and biological functions of the EAAEAE insert of GIF are poorly documented. NMR data showed that the backbone conformation of GIF is strikingly similar to that of mammalian MT1 and MT2, except for the fact that the EAAEAE acidic insert is unrestricted by the metal–thi- olate cluster and exhibits a dynamic loop conformation [9,10]. To explore the potential structural and func- tional consequences of the EAAEAE(55–60) insert in hGIF, a series of mutants at this site were designed, including (a) an EAAEAE(55–60)-deleted mutant (the D55-60 mutant), (b) an acidic residues-replaced mutant (the E55 ⁄ 58 ⁄ 60Q mutant protein) and (c) a helix- broken mutant (the E55D ⁄ A56G ⁄ A57G ⁄ E58D ⁄ A59G ⁄ E60D ⁄ A61G ⁄ E62D mutant protein) [16]. Neuronal bioassay results showed that the D55-60 mutant displayed a remarkable reduction in bioactivity compared with the wild-type hGIF, whereas the neuro- nal growth-inhibitory activities of other mutants designed at this site appeared to be similar to that of hGIF [16]. Biochemical studies showed that deletion of the EAAEAE insert greatly reduces the reaction rates of hGIF with 5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB) and S-nitrosocysteine (SNOC), and increases the stability of the metal–thiolate in the b-domain [11,16]. Then, we predicted the structure of intact hGIF and the D55-60 mutant. Unlike MT2, hGIF has a par- ticular strategy for its own domain–domain interaction and regulation mechanism. It was found that the Ser33- Cys38 and Lys31-Glu41 hydrogen bonds observed in hGIF fell apart in the D55-60 mutant, resulting in the movement of the a-domain away from the b-domain. These conformational alterations around the linker region of the D55-60 mutant also affected the interac- tion between Lys32 and the b-domain, leading to a con- formational change of the b-domain [16]. This agreed well with the results of previous SNOC and DTNB experiments [11,16]. Thus, it was concluded that dele- tion of the EAAEAE(55–60) insert in hGIF would make the A-cluster wrap more tightly and alter the domain–domain interactions in hGIF through intramo- lecular interactions, and eventually affect the structural and dynamic properties of the b-domain by domain– domain interactions, which would be a reason for the reduced bioactivity of the D55-60 mutant. The role of the linker It was found that the linker connecting the two domains of MTs is so conserved that in all mammalian MTs it exists as a KKS sequence (except for MT4, in which the conservative substitution linker RKS is found) (Fig. 1). Hence, we constructed three mutants of hGIF in the link region (the K31 ⁄ 32A mutant, the K31 ⁄ 32E mutant and the KKS-SP mutant) and attempted to explore the possible roles of the linker in the structure and function of GIF [24]. It was found that all three mutations reduce the stability of the b-domain and make it looser. These results undoubtedly reflect that the linker KKS(31–33) is more helpful in maintaining the stability of the metal–thiolate in the b-domain of hGIF. This was not surprising because our previous study demonstrated that Lys32 in the linker forms a hydrogen bond with Cys22 (2.92 A ˚ ) in the b-domain of hGIF. This hydrogen bond is believed to play an important role in the stability of the metal–thio- late cluster in the b-domain of hGIF [16]. Hence, when we changed the KKS linker to a linker with a different sequence, the hydrogen bond between Lys32 and Cys22 might disappear, thus decreasing the stability of the metal–thiolate clusters in the b-domain of hGIF. More significantly, changing KKS to SP also alters the general backbone conformation and metal–thiolate cluster geometry [24]. Interestingly, bioassay results showed a clear decrease in the bioactivity of the K31 ⁄ 32A and the K31 ⁄ 32E mutants, while the KKS-SP mutant showed a complete los of inhibitory activity [24]. Based on these results, it was proposed that the KKS linker is a crucial factor in modulating the stability and the solvent accessibility of the Cd 3 S 9 cluster in the b-domain through domain–domain interactions, and is thus indispensable for the biological activity of hGIF. The role of the acid–base catalysis site of hGIF It was reported that nitric oxide (NO) reacts with the thiolate group of MTs under pseudo-first-order condi- tions, leading to the release of zinc ions [25]. However, it was found that GIF is significantly more reactive than MT1 and MT2 towards S-nitrosothiols [26]. Chen et al. attributed the high activity of GIF towards SNOC to the unique acid–basic catalysis motif in the b-domain: KCE(21–23) [16]. To understand the role of the acid– basic catalysis motif in S-nitrosylation, we constructed an E23K mutant protein of hGIF by comparing the pri- mary sequence between hGIF and hMT1g [a human MT1 isoform where the segment is KCK(20–22)] [27]. Interestingly, it was found that the reaction of the E23K mutant with SNOC exhibits biphasic kinetics, and the reaction is much faster than that of hGIF at the initial step [27]. This result was not anticipated, indicating that the acid–base motif might not be the only factor Structure–reactivity–function study of GIF Z C. Ding et al. 2918 FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS contributing to the high activity of hGIF towards SNOC. Based on the results of CD spectral study, reac- tion with EDTA and pH titration, the structure and sta- bility of the metal–thiolate clusters of the E23K mutant, compared with those of hGIF, are not very different. The differences between hGIF and its E23K mutant lie in the SNOC reaction and solvent accessibility of the clusters. Compared with S-nitrosylation of hGIF, it is obvious that the E23K mutant, with more accessible clusters, is more reactive towards SNOC at the initial step, which makes us postulate that solvent accessibility of the clusters, besides acid–base catalysis, may be another important factor influencing S-nitrosylation of hGIF [27]. Unexpectedly, it was found that the neuronal growth- inhibitory activity of the E23K mutant is abolished completely [27]. As mentioned earlier, neither the over- all structure nor the stability of the metal–thiolate clus- ters of the E23K mutant were markedly different from those of wild-type hGIF. However, the SNOC reaction and NMR results clearly showed that Glu23 plays a spe- cific and important role in converting NO signals into zinc signals. These results indicate a connection of the role of this unique protein in zinc homeostasis with the NO signaling pathway. Based on these results, we sug- gest that mutation at Glu23 may alter the NO metabo- lism and ⁄ or affect zinc homeostasis in the brain, thus abolishing the neuronal growth-inhibitory activity [27]. Conclusion By studying a series of site-directed mutants of hGIF, it was suggested that the mechanism of the inhibitory activity of GIF is complex and is cooperatively regu- lated by multiple factors. The TCPCP motif is an important factor, which induces the characteristic con- formation in the N-terminus of GIF. Such a conforma- tion is indispensible for the neuronal growth-inhibitory activity of GIF. Other factors include solvent accessibil- ity and dynamic properties of the metal–thiolate cluster (especially the metal–thiolate cluster in the b-domain), which are closely associated with the mutual accessibil- ity of metal–thiolate clusters with biologically sensitive small molecules such as NO, thus influencing zinc homeostasis in the brain. Another factor identified is domain–domain interactions, which might play impor- tant roles in modulating the stability of the metal– thiolate cluster and the conformation of the b-domain. Comments The particular reactivity of GIF related to the specific metal–thiolate cluster has been reviewed by Peter Faller in the paper entitled ‘Reactivity and structure of metal-thiolate clusters in growth inhibitory factor’. Furthermore, Roger S. Chung and coworkers summa- rized current understandings on the biological func- tions of GIF in the article entitled ‘A current evaluation of the biological function of growth inhibi- tory factor in the injured and neurodegenerative brain’. Recently, the putative key role of b-amyloid (Ab) peptide in the pathogenesis of AD led to a promising outlook for the treatment of AD. However, the AN1792 trail report on AD patients showed that ‘although immunization with Ab 42 (AN1792) resulted in clearance of amyloid plaques in patients with Alzheimer’s disease, this clearance did not prevent progressive neuro-degeneration’ [28]. It was also reported that during the formation of Ab–protein aggregates, free radicals were generated by redox metal ion catalyses, which damage neuron cells and lead to their death. Therefore, it is highly probable that one of the causes of AD could be the distur- bance of essential metal homeostasis in the brain, as the brain consumes almost one-quarter of the total oxygen in humans, and also Cu, Fe and Zn are heavily deposited in the senile plaque and neurofibril tangle. Any disorder or malfunction in the metallo- proteins and metalloenzymes that maintain and regu- late the homeostasis of essential metal ions in brain could lead to neuron-degenerative diseases. Surely, MTs are a family of novel metalloproteins. 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MINIREVIEW Neuronal growth-inhibitory factor (metallothionein-3): structure–function relationships Zhi-Chun Ding*, Feng-Yun. 5,5¢-dithiobis-(1-nitrobenzoic acid); GIF, neuronal growth-inhibitory factor; hGIF, human neuronal growth-inhibitory factor; MT, metallothionein; NO, nitric