Chemistry Reference
In-Depth Information
used in initial structure generation to avoid bias, especially during the automated
assignment procedure in CYANA [
39
]; instead, metal ligand restraints are
incorporated in the last step of structural calculation as well as in the refinement
stage [
40
,
41
].
The application of NMR spectroscopy in protein structure determination actually
started with a small metalloprotein, metallothionine (MT) [
42
]. Metallothionines are
a class of low molecular weight (typically 6-7 kDa) cysteine-rich proteins. The
proteins lack a well-defined secondary structure and their folding is dictated mostly
by a clustered network of cysteine residues and metal ions usually represented by Zn
2
+
,Cu
+
,andCd
2+
[
43
,
44
]. Since the first solution of the structure of rabbit liver
Cd
7
MT2 [
42
], numerous three dimensional structures of metallothiones from differ-
ent isoforms (MT1/MT2/MT3) or different species such as blue crab and mammalian
(rabbit, rat, and human) have been resolved by NMR spectroscopy [
45
]withonly
one structure (rat liver Cd
5
Zn
2
MT2) determined by X-ray crystallography [
46
]. The
protein consists of two dynamic metal-thiolate clusters folded into two domains
(
) and the structural mobility of the protein makes it difficult to be crystallized.
The metal cluster restraints, e.g., Cd-S bond lengths, as well as Cd-S-Cd, S-Cd-S,
and CysC
b
-S-Cd bond angles from the X-ray crystal structures of model cadmium
complexes and rat liver Cd
5
Zn
2
MT2 were often incorporated with other distance
and angle restraints in structure calculation. Recently, a new member of metallo-
thionine MT3 with the conserved CPCP motif in the N-termini has been involved in
the growth inhibitory activity and is down-regulated in the brain of Alzheimer's
patients [
47
]. The solution structures of both human [
27
] and mouse MT3 [
48
]
resolved by NMR spectroscopy for the C-terminal
a
,
b
-domain, Fig.
1a
, revealed
a similar Cd
4
Cys
11
cluster as well as very similar tertiary folds to MT1/2. However,
a loop in the acidic hexapeptide insertion is found and is slightly longer in human
MT3 than in mouse MT3. The first solution structure of Cd
7
MT-nc of the Antarctic
fish
Notothenia coriiceps
was also determined [
28
]. The position of the ninth cysteine
of Cd
7
MT-nc is different frommammalian MT which results in a structural change of
the domain, in particular in the orientation of the loop (Lys50-Thr53), Fig.
1b
,andin
turn a different charge distribution with respect to mammalian MT [
28
]. Interestingly,
an intriguing class of histidine-containing metallothionines has also been identified in
fungi and bacterial [
49
]. The histidine residue has been thought to be able to modulate
zinc affinity and reactivity. Solution structure of one of this class of MTs, Zn
4
SmtA
from cyanobacterium
Synechococcus
PCC 7942 was determined [
41
], Fig.
1c
,reveal-
ing a Zn
4
Cys
9
His
2
cluster with a topology similar to that of the Zn
4
Cys
11
cluster
of the
a
-domain of mammalian MT. However, the two ZnCysHis sites and one
ZnCys
4
site readily exchange Zn
2+
for exogenous Cd
2+
. Moreover, SmtA contains
ashort
a
-sheets surround the inert zinc site, which
resemble zinc finger portions of GATA and LIM proteins. Such a structure of SmtA
probably produces its function of specific protein and/or DNA recognition [
41
].
NMR spectroscopy makes an enormous contribution to structural biology of
metalloproteins, particularly in zinc-binding proteins. Zinc, the second most abundant
metal found in eukaryotic organisms, plays important catalytic and structural roles
in a variety of biological processes. Binding of zinc is able to stabilize the folded
a
-helix and two small antiparallel
b
