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doi:10.1016/j.jmb.2008.04.004
Zn2+, an element that is essential to all life forms, can play a catalytic or a
solely structural role. Previous works have shown that Zn2+ binds preferentially to water molecules and His in catalytic sites, but to Cys instructural
sites, but the molecular basis for the observed ligand preference is unclear.
Here, we show that the different Zn2+ roles are also reflected in the different
bond distances to Zn2+ in structural and catalytic sites. We reveal the
physical basis for the observed differences between structural and catalytic
Zn sites: In most catalytic sites, water is found bound to Zn2+ as it transfers the
least charge to Zn2+ and is less bulky compared to the protein ligands,
enabling Zn2+ to serve as a Lewis acid in catalysis. In most structural sites,
however, 2 Cys are found bound to Zn2+, as Cys transfers the most
charge to Zn2+ and reduces the Zn charge to such an extent that Zn2+ can no
longer act as a Lewis acid; furthermore, steric repulsion among the bulky Cys
(S) prevents Zn2+ from accommodating another ligand. Based on the
observed ligand preference and Znligand distance differences between
structural and catalytic Zn sites, we present a simple method for distinguishing the two types of sites and for verifying the catalytic role of Zn2+.
Finally, we discuss how the physical bases revealed aid in designing potential drug molecules that target Zn proteins.
2008 Elsevier Ltd. All rights reserved
Edited by D. Case
Introduction
One of the most important trace metal ions in living
organisms is Zn2+, an essential cofactor in many metabolic enzymes and regulatory proteins.17 Zn2+ can
play a catalytic role (catZn) or a solely structural role
(strZn) in proteins. In catZn sites, Zn2+ can act as a
Lewis acid (i.e., an electron-pair acceptor or an emptyorbital donor) and facilitate Zn-bound water to ionize
to a nucleophilic hydroxide in enzymes such as
*Corresponding author. Institute of Biomedical Sciences,
Academia Sinica, Taipei 115, Taiwan. E-mail address:
carmay@gate.sinica.edu.tw.
Abbreviations used: catZn, catalytic Zn; strZn,
structural Zn; HCA2, human carbonic anhydrase II; 3D,
three-dimensional; PDB, Protein Data Bank; ColE7, colicin
E7; ColE9, colicin E9; DNase, deoxyribonuclease; CSD,
Cambridge Structure Database; CN, coordination
number; BVS, bondvalence sum; HIV, human
immunodeficiency virus; IE, ionization energy; EA,
electron affinity.
0022-2836/$ - see front matter 2008 Elsevier Ltd. All rights reserved
546
This suggests that the protein environment, in addition to Zn ligands, determines Zn's role in certain
proteins sharing the same metal complex.
The question of whether Zn2+ plays primarily a
structural or a catalytic role may still be uncertain
even when the respective protein structure is known.
For instance, it was unclear whether Zn2+ plays a
purely structural and/or a catalytic role in colicin E7
(ColE7) and colicin E9 (ColE9), which are protein
toxins made by bacteria to kill other bacteria via an
endonuclease domain.15 The X-ray structures of
ColE7 and ColE9 deoxyribonuclease (DNase) domains (1m08 and 1fsj) show Zn2+ tetrahedrally coordinated to three His residues and a phosphate
oxygen molecule. The Zn2+ in ColE9 has been hypothesized to play a structural role based on experimental results showing the Zn-bound ColE9 DNase
domain to be more thermally stable than the apoprotein and to be active with Ni2+, Co2+, and Mg2+,
but inactive with Zn2+.16 However, the Zn2+ in
ColE7, which shares a ~ 70% sequence identity with
ColE9, has been postulated to play a catalytic role
based on experimental results showing the Zn2+ to
be dispensable for DNA binding but essential for
DNA hydrolysis.17
The debatable role of Zn2+ in bacterial endonuclease enzymes, the Zn ligand preference observed in
cat
Zn and strZn sites, and the role of Zn2+ as a Lewis
acid in catZn sites raise several intriguing questions
that, to the best of our knowledge, have not been
addressed in previous works: (1) Are the different
Zn2+ roles reflected in different distances between
Zn2+ and a given ligand (L) atom in the two types of
sites? (2) If so, what is the physical basis for the observed differences in ligand preference and ZnL
distances between catZn and strZn sites (e.g., why do
most strZn sites strongly prefer Cys to other amino
acid ligands and how does this affect the metal's
properties/function?)? (3) Can the feature(s) governing the different Zn2+ roles be exploited to distinguish
cat
Zn from strZn sites, especially in 3D structures
where the Zn-bound water position is unknown/
Results
ZnL distances in
str
Zn and
cat
Zn sites
Table 1. Average distances () between Zn2+ and its coordinating atoms in PDB structures, as compared to those in CSD
structures and BVS distances
Zn type
T4 CSD
T4 BVS
T4 Strc
T4 Cat
T5 CSD
T5 BVS
T5 Catg
T6 CSD
T6 BVS
T6 Cat
ZnS
2.32 0.05
2.35
2.33 0.09
2.38 0.18
2.42 0.13
2.43
ZnO
ZnN
(246)
(137)
(16)
(46)
(2)
2.04 0.04
2.03
2.05 0.16
2.16 0.12
2.10 0.08
2.11
2.08 0.07
2.16 0.05
2.18
2.10 0.10
(603)
(36)
(64)
(38)
(38)
(409)
(7)
1.97 0.03
1.96
2.09 0.17
2.09 0.04
2.04
2.16 0.19
2.10 0.06
2.11
2.19 0.17
ZnOwater
(109)
(2)
(36)
(12)
(46)
(103)
(11)
2.00 0.04
1.96
2.28 0.23
2.01 0.04
2.04
2.24 0.25
2.11 0.06
2.11
2.27 0.11
(21)
(1)
(27)
(5)
(24)
(274)
(6)
The number after the distance is the standard deviation, and the number in parentheses is the number of observations. When the number
of observations is b 5, the mean distance is not given as it is statistically unreliable.
CSD: distances derived from CSD structures (Kuppuraj and Lim, in preparation); BVS: distances calculated using Eq. 3 (see Materials
and Methods); Strc: distances derived from the strZn sites in Supplementary Table S1; Cat: distances derived from the catZn sites in
Supplementary Table S2.
reflecting perhaps the need for Zn2+ to adopt different CNs during an enzymatic reaction. In strZn sites,
however, Zn2+ is tetracoordinated: no T5 site and one
T6 site (1enr) was found. Hence, only the ZnL
distances in T4 strZn and catZn sites were compared.
The different roles of Zn2+ are reflected not only in
the different metal CNs but also in the different ZnL
distances in strZn and catZn sites. In strZn sites, the
ZnN and ZnS distances (2.05 and 2.33) are
similar to those found in CSD structures (2.04 and
2.32). In catZn sites, however, the ZnN distance
(2.16) is longer than that in strZn sites; the ZnO
and ZnOwater distances (which cannot be compared with those in strZn sites due to lack of statistics)
are longer than those in the T4 CSD structures. The
longer ZnOwater distance in catZn sites relative to
CSD structures has been attributed to hydrogenbonding interactions with the Zn-bound water in the
enzyme, pulling it away from Zn2+.10
Effect of the Zn ligands on Zns Lewis acidity
To account for the observed preference of Cys to His
in strZn sites and vice versa in catZn sites, the electronegativity () of Zn complexes modeling common
str
Zn and catZn cores (Fig. 1) was evaluated (see Materials and Methods). The values in Table 2 provide
a measure of Zn's ability to act as a Lewis acid, since
only Zn2+ can accept electrons in the metal complex.
As the number of Zn-bound Cys decreases, the of
the complex increases (from 2.50 to 6.76eV), implying that electrons will flow more easily from an
electron donor (e.g., substrate) to catZn cores than to
structural ones. Thus, Zn's Lewis acidity can be modulated by its bound ligands, in particular by the
number of Zn-bound Cys, and Zn2+ would be a
better Lewis acid in catZn sites than in strZn sites.
To provide a physical basis for the greater values
of catZn sites, as compared to strZn sites, the net charge
transferred by all the ligands to Zn2+ was computed
from the Zn's charge. As the of the complex
increases, the net charge transferred by the ligands to
Zn2+ decreases (Table 2). Comparison of the charge
transfer between [Zn(CH3S)4]2 and [Zn(CH3S)3(ImH)],
between [Zn(CH3S)3(ImH)] and [Zn(CH3S)2(ImH)2]0,
or between [Zn(CH3S)2(ImH)2]0 and [Zn(OH)(ImH)3]+
shows that a negatively charged ligand transfers more
charge to Zn2+ than a neutral one. Notably, even
when the net charge of the complex is the same, the
charge transfer in the [Zn(CH3S)2(ImH)2]0 model
structural core (0.68e) is greater than that in the [Zn
(OH)(HCOO)(ImH)2]0 model catalytic core (0.43e),
implying that Cys transfers more charge to Zn2+
than Asp /Glu or OH. Indeed, Cys has been
shown to transfer the most charge to Zn2+, while
water has been shown to transfer the least charge.21
Effect of the ZnL changes on Zns Lewis acidity
To evaluate whether the observed differences in
the ZnL distances affect Zn's Lewis acidity, the
+
condensed Fukui functions at Zn2+ (f Zn
), which
reflect Zn's electron-acceptor ability, were computed
547
from the fully optimized geometry of the metal complex and from the constrained geometry of the respective complex, with the ZnL distances fixed to
those in b 2.5- PDB and CSD structures, as described in Materials and Methods (see Supplementary
Table S3). The PDB-constrained geometries reflect
the effect of the protein matrix on the isolated Zn
+
values derived from
complex structures. The f Zn
various conformations of the same metal complex
and metal CN were compared: Those derived from
the PDB-constrained T4 geometries were compared
+
derived from the fully optimized geowith the f Zn
metry (whose ZnL distances were similar to those in
the CSD structures). Those derived from the PDBconstrained T5 geometries were compared with the
+
derived from the respective CSD-constrained T5
f Zn
geometry, as full optimization would yield a tetra+
values for the
hedral complex. The relative f Zn
str
cat
common Zn and Zn cores are depicted in Fig. 2,
along with the sum of the deviations of the ZnL
distances in the PDB site (diPDB ) from those in the
fully optimized or CSD-constrained reference structure (diREF); (diPDB diREF) values that are b 1 are in
gray, while those that are 1 are in black.
The net bond length changes (diPDB diREF) in strZn
sites containing 2 Cys, where Zn2+ is already a
poor Lewis acid, negligibly affect Zn's electron-acceptor ability, but those in catZn sites alter Zn's Lewis
acidity. In strZn sites, the net bond length changes are
+
changes are
as large as 0.53, but the respective f Zn
+
negligible (f Zn 0.009). This trend is found in the
three most common strZn sites, regardless of the metal
complex's net charge. Thus, relative to the isolated Zn
complex, the different bond lengths in the strZn sites
in protein structures negligibly affect Zn's Lewis
acidity. In contrast, in catZn cores, the net bond length
+
values are at
changes are 0.21.6, while the f Zn
least an order of magnitude greater than those in strZn
sites, implying that the ZnL distance changes can
modulate Zn's Lewis acidity.
Distinguishing strZn and
ligand preference
cat
Zn sites based on
548
Fig. 1. The S-VWN/631+G(d) fully optimized structures modeling common structural and catalytic Zn cores.
549
Model Zn complex
2
[Zn Cys4]
[Zn Cys3 His]
[Zn Cys2 His2]0
[Zn Cys Asp/Glu
His H2O]0
[Zn OH Asp/Glu
His2]0
[Zn OH His3] +
[Zn (CH3S)4]
[Zn (CH3S)3 ImH]
[Zn (CH3S)2 (ImH)2]0
[Zn CH3S HCOO
ImH H2O]0
[Zn OH HCOO (ImH)2]0
[Zn OH (ImH)3] +
(eV)a
CT (e)b
2.50
0.48
3.29
3.64
0.81
0.76
0.68
0.58
4.11
0.43
6.76
0.40
cat
550
Discussion
Whereas the different roles of Zn2+ have been found
in previous works1,1012 to be reflected in the different
Fig. 4. Distinguishing strZn and catZn sites based on ligand preference. (a) Flowchart showing how catZn sites can be
distinguished from structural ones by counting the number of Zn-bound Cys and water ligands. (b) The distribution of
predicted strZn and catZn sites.
551
PDB code/
chain
1fsj/B
1fsj/C
1fsj/D
1fsj/E
1m08/A
1m08/B
7cei
1mz8/B
1mz8/D
a
b
Molecule(s)
ColE9
2.08
2.06
2.08
2.11
ColE7
1.86
2.05
ColE7 + Im7 2.06
ColE7 + Im7 1.98
2.09
2.08
2.09
2.17
2.14
2.10
2.08
1.92
2.22
2.10
2.07
2.08
2.08
2.07
2.03
2.09
2.57
2.07
2.15
1.94
2.00
1.95
1.99
1.79
1.90
2.16b
2.04
2.07
1.83
1.75
1.71
1.67
2.48
1.90
1.52
1.71
1.56
cat
Zn and
str
Zn sites
552
that the mean ZnN, ZnOwater, ZnO, and ZnS distances do not exceed 2.16, 2.11, 2.10, and 2.42, respectively
(Table 1). On the other hand, analyses of b 2.5- X-ray
structures of the same protein (HCA2) have shown that a
given ZnL distance could vary by as much as 0.4. This
uncertainty would be larger for exchanging water ligands.
Thus, to account for the lower resolution of the PDB structures, as compared to the CSD structures, 0.4 was added
to the mean ZnN, ZnO, and ZnS CSD distances, while
0.8 was added to the ZnOwater CSD distance to locate
the first-shell Zn ligands in the PDB structures. For a given
metal CN, if the ZnOwater distance in the PDB structure is
below 1 S.D. of the respective ZnOwater CSD distance
(i.e., less than 1.96, 1.97, and 2.05 for T4, T5, and T6 sites,
respectively), it is treated as a ZnO distance.
fZn
qZn N1qZn N
Models used
We modeled the most common strZn ([Zn(Cys)4]2, [Zn
(Cys)3His], and [Zn(Cys)2(His)2]0) and catZn ([Zn(Cys)(Asp/
Glu)(His)(H2O)]0, [Zn(OH)(Asp/Glu)(His)2]0, and [Zn(OH)
(His)3]+) cores in proteins (Fig. 1). In the latter two complexes,
the Zn-bound water was assumed to be deprotonated in the
catalytically active state, in accordance with the measured
pKa values of the Zn-bound water in HCA28 and carboxypeptidase A.9 The side chains of Cys, His, and Asp/Glu
were modeled by methyl thiolate (CH3S), imidazole (ImH),
and formate (HCOO), respectively.
Geometry optimization
The model strZn and catZn cores were fully optimized or
constrained optimized by fixing only the ZnL distances to
the values observed in the b 2.5- PDB structures (Supplementary Table S3); the overall fully optimized and constrained optimized geometries are similar on visual
inspection. The S-VWN functional set and the 631+G(d)
basis set had been found adequate for geometry optimization of Zn complexes containing methyl thiolate, imidazole,
and/or formate ligands. Since S-VWN/631+G* reproduces
the experimentally observed ZnL distances in related Zn
complexes better than other functional set/basis set
combinations,2931 it was used to optimize the geometries
of the Zn complexes using the Gaussian 03 program.32
Computing the global reactivity descriptor
The electronegativity was estimated by averaging the
molecule's ionization energy (IE) and electron affinity (EA),33
IE EA
vi
2
Acknowledgements
We thank H.S. Yuan, T. Dudev, T.W. Chang, I.
Tunell, B. Chen, and G. Kuppuraj for helpful discussions. This work was supported by the National
Science Council, Taiwan (contract no. 95-2113-M001-001).
Supplementary Data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2008.04.004
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