Tài liệu Báo cáo khoa học: Neuronal growth-inhibitory factor (metallothionein-3): structure–function relationships docx

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:
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.
As I said in 1994 ‘metallothionein is a fascinating mol-
ecule and a real beauty of nature. It is a small protein,
but it is like a drop of morning dew reflecting from
every aspect the magnificent color of magical biologi-
cal molecules. When we gradually understand more
chemistry and biology about it, it would not be sur-
prising to find that nature produced such an elegant
molecular device to perform a variety of biological
functions.’
Acknowledgment
The authors are grateful for financial support from the
National Natural Science Foundation of China.
References
1 Margoshes M & Vallee BL (1957) A cadmium protein
from equine kidney cortex. J Am Chem Soc 79, 4813–

4814.
2 Binz PA & Kagi JHR (1999) Metallothionein IV,
Metallothionein: Molecular Evolution and Classification.
Birkhause, Baser.
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 2919
3 Palmiter RD (1987) Molecular biology of metallothion-
ein gene expression. Experientia Suppl 52, 63–80.
4 Uchida Y & Tomonaga M (1989) Neurotrophic action
of Alzheimer’s disease brain extract is due to the loss of
inhibitory factors for survival and neurite formation of
cerebral cortical neurons. Brain Res 481, 190–193.
5 Uchida Y & Ihara Y (1995) The N-terminal portion of
growth inhibitory factor is sufficient for biological activ-
ity. J Biol Chem 270, 3365–3369.
6 Sewell AK, Jensen LT, Erickson JC, Palmiter RD &
Winge DR (1995) Bioactivity of metallothionein-3 cor-
relates with its novel beta domain sequence rather than
metal binding properties. Biochemistry 34, 4740–4747.
7 Robbins AH, McRee DE, Williamson M, Collett SA,
Xuong NH, Furey WF, Wang BC & Stout CD (1991)
Refined crystal structure of Cd, Zn metallothionein at
2.0 A resolution. J Mol Biol 221, 1269–1293.
8 Braun W, Vasak M, Robbins AH, Stout CD, Wagner
G, Kagi JH & Wuthrich K (1992) Comparison of the
NMR solution structure and the x-ray crystal structure
of rat metallothionein-2. Proc Natl Acad Sci USA 89,
10124–10128.
9 Wang H, Zhang Q, Cai B, Li H, Sze KH, Huang ZX,
Wu HM & Sun H (2006) Solution structure and

dynamics of human metallothionein-3 (MT-3). FEBS
Lett 580, 795–800.
10 Oz G, Zangger K & Armitage IM (2001) Three-dimen-
sional structure and dynamics of a brain specific growth
inhibitory factor: metallothionein-3Biochemistry 40,
11433–11441.
11 Zheng Q, Yang WM, Yu WH, Cai B, Teng XC, Xie Y,
Sun HZ, Zhang MJ & Huang ZX (2003) The effect of
the EAAEAE insert on the property of human metallo-
thionein-3. Protein Eng 16, 865–870.
12 Ni FY, Cai B, Ding ZC, Zheng F, Zhang MJ, Wu HM,
Sun HZ & Huang ZX (2007) Structural prediction of
the beta-domain of metallothionein-3 by molecular
dynamics simulation. Proteins 68, 255–266.
13 Yu H, Chen JK, Feng S, Dalgarno DC, Brauer AW
& Schreiber SL (1994) Structural basis for the binding
of proline-rich peptides to SH3 domains. Cell 76,
933–945.
14 Williamson MP (1994) The structure and function of
proline-rich regions in proteins. Biochem J 297 (Pt 2),
249–260.
15 Cai B, Zheng Q, Teng XC, Chen D, Wang Y, Wang
KQ, Zhou GM, Xie Y, Zhang MJ, Sun HZ et al.
(2006) The role of Thr5 in human neuron growth inhib-
itory factor. J Biol Inorg Chem 11, 476–482.
16 Cai B, Ding ZC, Zhang Q, Ni FY, Wang H, Zheng Q,
Wang Y, Zhou GM, Wang KQ, Sun HZ et al. (2009)
The structural and biological significance of the
EAAEAE insert in the alpha-domain of human
neuronal growth inhibitory factor. FEBS J 276, 3547–

3558.
17 Hasler DW, Jensen LT, Zerbe O, Winge DR & Vasak
M (2000) Effect of the two conserved prolines of human
growth inhibitory factor (metallothionein-3) on its bio-
logical activity and structure fluctuation: comparison
with a mutant protein. Biochemistry 39, 14567–14575.
18 Romero-Isart N, Jensen LT, Zerbe O, Winge DR &
Vasak M (2002) Engineering of metallothionein-3
neuroinhibitory activity into the inactive isoform
metallothionein-1. J Biol Chem 277, 37023–37028.
19 Chung RS, Holloway AF, Eckhardt BL, Harris JA,
Vickers JC, Chuah MI & West AK (2002) Sheep have
an unusual variant of the brain-specific metallothionein,
metallothionein-III. Biochem J 365 , 323–328.
20 Faller P, Hasler DW, Zerbe O, Klauser S, Winge DR &
Vasak M (1999) Evidence for a dynamic structure of
human neuronal growth inhibitory factor and for major
rearrangements of its metal-thiolate clusters. Biochemis-
try 38, 10158–10167.
21 Faller P & Vasak M (1997) Distinct metal-thiolate clus-
ters in the N-terminal domain of neuronal growth
inhibitory factor. Biochemistry 36, 13341–13348.
22 Jiang LJ, Vasak M, Vallee BL & Maret W (2000) Zinc
transfer potentials of the alpha - and beta-clusters of
metallothionein are affected by domain interactions in
the whole molecule. Proc Natl Acad Sci USA 97, 2503–
2508.
23 Ding ZC, Zheng Q, Cai B, Yu WH, Teng XC, Wang
Y, Zhou GM, Wu HM, Sun HZ, Zhang MJ et al.
(2007) Effect of alpha-domain substitution on the struc-

ture, property and function of human neuronal growth
inhibitory factor. J Biol Inorg Chem 12, 1173–1179.
24 Ding ZC, Teng XC, Zheng Q, Ni FY, Cai B, Wang Y,
Zhou GM, Sun HZ, Tan XS & Huang ZX (2009) Impor-
tant roles of the conserved linker-KKS in human neuro-
nal growth inhibitory factor. Biometals 22, 817–826.
25 Aravindakumar CT, Ceulemans J & De Ley M (1999)
Nitric oxide induces Zn
2+
release from metallothionein
by destroying zinc-sulphur clusters without concomitant
formation of S-nitrosothiol. Biochem J 344 (Pt 1),
253–258.
26 Chen Y, Irie Y, Keung WM & Maret W (2002)
S-nitrosothiols react preferentially with zinc thiolate
clusters of metallothionein III through transnitrosation.
Biochemistry 41, 8360–8367.
27 Ding ZC, Teng XC, Cai B, Wang H, Zheng Q, Wang
Y, Zhou GM, Zhang MJ, Wu HM, Sun HZ et al.
(2006) Mutation at Glu23 eliminates the neuron growth
inhibitory activity of human metallothionein-3. Biochem
Biophys Res Commun 349, 674–682.
28 Holmes C, Boche D, Wilkinson D, Yadegarfar G,
Hopkins V, Bayer A, Jones RW, Bullock R, Love S,
Neal JW et al. (2008) Long-term effects of Ab
42
immunisation in Alzheimer’s disease: follow-up of a
randomised, placebo-controlled phase I trial. Lancet
372, 216–223.
Structure–reactivity–function study of GIF Z C. Ding et al.

2920 FEBS Journal 277 (2010) 2912–2920 ª 2010 The Authors Journal compilation ª 2010 FEBS