Tài liệu Báo cáo khoa học: The solution structure of reduced dimeric copper zinc superoxide dismutase doc

The solution structure of reduced dimeric copper zinc superoxide
dismutase
The structural effects of dimerization
Lucia Banci, Ivano Bertini, Fiorenza Cramaro, Rebecca Del Conte and Maria Silvia Viezzoli
Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, Italy
The solution structure of homodimeric Cu
2
Zn
2
superoxide
dismutase (SOD) of 306 aminoacids was d etermined on a
13
C,
15
N a nd 70%
2
H l abeled sample. Two-thousand e ight-
hundred and five meaningful NOEs were used, of which 96
intersubunit, and 115 dihedral angles provided a f amily of 30
conformers with an rmsd from the average of 0.78 ± 0.11
and 1.15 ± 0.09 A
˚
for the backbone and heavy atoms,
respectively. When the rmsd is calculated for each subunit,
the values drop t o 0.65 ± 0.09 and 1.08 ± 0.11 A
˚
for the
backbone and heavy atoms, respectively.
The two subunits are identical on the NMR time scale, at
variance with the X-ray structures that show structural dif-
ferences between the two subunits as well as between dif-

ferent molecules in the unit cell. The elements of secondary
structure, i.e. eight b sheets, are the same as in the X-ray
structures and are well defined. The odd loops (I, III and V)
are well resolved as well as loop II located at the subunit
interface. On the c ontrary, l oops IV and V I show some
disorder. The residues of the active cavity are well defined
whereas within th e various subunits of the X-ray structure
some are disordered or display different orientation in dif-
ferent X-ray structure determinations. T he copper(I) ion and
its ligands are well defined. This structure t hus represents a
well defined model in solution relevant for structure–func-
tion analysis of the protein. T he comparison between the
solution structure of monomeric mutants and the present
structure shows that the subunit–subunit interactions in-
crease the order in loop II. This has the consequences of
inducing the structural and d ynamic properties that a re
optimal for the enzymatic function of the wild-type enzyme.
The regions 37–43 and 89–95, constituting loops III and V
and the initial p art of t he b barrel and s howing several
mutations in familial amyotrophis lateral sclerosis (FALS)-
related proteins have a quite extensive network of H-bonds
that may account for t heir low mobility. Finally, t he con-
formation of the key Arg143 residue is compared to that in
the other dimeric and monomeric structures as well as in the
recently reported structure of the CCS–superoxide dismu-
tase (SOD) complex.
Keywords: superoxide dismutase; solution structure; dimeric
protein; NMR; FALS.
Cu
2

Zn
2
SOD is a well known homodimeric enzyme of
32 000 Da that catalyzes the dismutation of the superoxide
radical to h ydrogen p eroxide a nd oxygen through a two s tep
reaction [1–5]:
Cu
2þ
þ O
À
2
! Cu
þ
þ O
2
Cu
þ
þ O
À
2
! Cu
2þ
O
2À
2
ÀÁ
!
2H
þ
Cu

2þ
þ H
2
O
2
The active site of each subunit contains both a zinc and a
copper ion, the latter being the site of the reaction. Copper
occurs in the oxidized and in th e r educed state, both of
which are necessary for the function. The X-ray structure of
the oxidized form has been available since 1982 for the
bovine enzyme [6,7] and several other structures have
become available [8–18]. Reduced state structures are also
available although the picture is less clear-cut around
the copper-binding site [19–21]. Certainties on the protona-
tion of His63, which b ridges Cu an d Zn in th e oxidized form
but is protonated in the reduced form, come from
1
HNMR
studies [22–25]. Eventually, monomeric forms were
obtained through site-specific mutagenesis and t he NMR
solution structure [26,27] as well as the crystal structure [28]
of the reduced form were re ported. Also the b ackbone
mobility of the monomeric state was investigated and
compared with that of the dimeric species and it was
concluded t hat, as far as motions in the ps to ns timescale
are c oncerned, t he region c onsisting of residues 131–142,
which forms one side of the a ctive site channel, is less mobile
in the monomeric mutant than in the dimeric wild-type
protein; structu ral fluctuations in this region have been
suggested to play a role in assisting the superoxide anion in

sliding towards the active site [29,30]. Moreover, the regions
consisting of residues 47–59, 76–86 and 151–153, which are
Correspondence to I. Bertini, Department of Chemistry and Centro
Risonanze Magnetiche, University of Florence, Via Luigi Sacconi 6,
50019 Sesto Fiorentino, Italy.
Fax: + 39 055 4574271, Tel.: + 39 055 4574272,
E-mail: fi.it
Abbreviations: SOD, superoxide dismutase; Q133M2SOD, F50E/
G51E/E133Q monomeric mutant superoxide dismutase; FALS,
familial amyotrophis lateral sclerosis; M4SOD, F50E/G51E/V148K/
I151K monomeric mutant superoxide dismutase; CCS, yeast copper
chaperone for superoxide dismutase; TPPI, time proportional phase
increments.
Note: The PDB ID code for the solution structure of homodimeric
Cu
2
Zn
2
superoxid e dismutase is 1L3N.
(Received 5 October 2001, revised 4 February 2002, accepted 16
February 2002)
Eur. J. Biochem. 269, 1905–1915 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02840.x
located at the subunit–subunit interface, were found to be
more rigid in the dimer. This behavior was rationalized by
considering that the presence of the second subunit
produces residue–residue interactions, thus reducing their
motions [30]. Also on the ms to ls time range, the subunit–
subunit interface displays increased m obility in the mono-
meric state with respect to the dimeric one. In particular,
conformational equilibria were observed for residues

around Cys57 a nd Cys146. T he former residue forms an
H-bond with the guanidinium group of Arg143 [31], which
is located in t he active site channel pointing towards the
copper ion and whose side-chain orientation is optimized
for correctly orienting the incoming superoxide an ion for
the electron transfer process. The equilibrium between
multiple conformations for this group and a different
average structural orientation does not allow Arg143 to
assume the optimal orientation for the enzymatic reaction
[26,27,30]. T his c ould a ccount for the reduced enzymatic
rates of the artificial monomeric species.
The X-ray structures of the dimeric wild-type form of this
protein show different structural details in the active site,
both between the two subunits of the same molecule and
among crystallographically independent subunits. This
holds also for a number of loops [32]. The question of
why SOD is a dimer and whether there is a cooperativity or
anticooperativity between the two subunits in the physio-
logical picture has never been completely solved.
In this context we decided to solve the solution structure
of the reduced dimeric protein (by using a classical NMR
approach) in order to compare the solution structures of the
monomeric and dimeric species as well as the solution and
the crystal structures. The aim is to classify the effects of
dimerization on the structural details.
MATERIALS AND METHODS
Sample preparation
Dimeric human SOD was expressed i n Escherichia coli
TOPP1 strain (Stratagene). The
15

Nand
15
N,
13
C,
2
H
labeled proteins were obtained b y growing the cells in
minimal m edium (M9) a s previously reported [30]. T he
samples were isolated and purified according to previously
published p rotocols [33]. The triple labeled dimeric SOD
contained about 70%
2
H. Reduction of the copper ion was
achieved by addition of sodium isoascorbate to a final
concentration of about 4–6 m
M
,in20 m
M
phosphate buffer
at pH 5.0 u nder anaerobic conditions. The NMR samples
had a concentration of about 2 m
M
in dimeric protein and
contained 10% D
2
O for the lock signal.
NMR experiments
The NMR experiments were recorded on Bruker Avance
800, 700 and 600 spectrometers operating at 18.7, 16.4 and

14.1 T, re spectively .
The assignment of the back bone is already available [30].
For the assignm ent of side chains H(C)CH-TOCSY, a t
600MHz,and(H)CCH-TOCSY,at800MHz[34],were
performed, using 1024 (
1
H) · 112 (
13
C) · 256 (
1
H) data
points and spectral windows of 9258 Hz (
1
H) · 11 184 Hz
(
13
C) · 9258 Hz (
1
H) and 1024 (
1
H) · 128 (
13
C) · 280
(
13
C) data points with spectral windows of 12 019 Hz
(
1
H) · 16 667 Hz (
13

C) · 16 667 Hz (
13
C), respectively. A
15
N-NOESY-HSQC and a
13
C-NOESY-HSQC [35] were
collected at 800 MHz to obtain dipolar connectivities; a
HNHA [36], at 700 MHz, and HNHB [37], at 800 MHz,
experiments, were performed to determine the
3
J
HNHa
coupling constants and additional constraints for the v
1
torsion angles as well as stereospecific assignments for the
Hb protons. The
15
N-NOESY-HSQC was recorded with
spectral windows of 9569 Hz (
1
H) · 2989 Hz (
15
N) ·
9569 Hz (
1
H) for 2048 (
1
H) · 88 (
15

N) · 29 6 (
1
H) data
points. The
13
C-NOESY-HSQC was acquired with 1024
(
1
H) · 112 (
13
C) · 256 (
1
H) data points with s pectral
windows of 9615 Hz (
1
H) · 19 230 Hz (
13
C) · 9615 Hz
(
1
H). For both experiments the m ixing time was 130 ms.
The three-dimensional HNHA experiment was carried o ut
using s pectral windows of 9124 Hz (
1
H) · 9124 Hz
(
1
H) · 3125 Hz (
15
N) for 1024 (

1
H) · 128 (
1
H) · 32 (
15
N)
data points, the three-dimensional HNHB and the two-
dimensional reference experiments were c arried out u sing
spectral windows of 11 160 Hz (
1
H) · 3244 Hz (
15
N) ·
11 160 Hz (
1
H) for 1024 (
1
H) · 48 (
15
N) · 12 8 (
1
H) and
11 160 Hz (
1
H) · 3244 Hz (
15
N) for 1024 (
1
H) · 256 (
15

N)
data points, respectively.
These experiments were collected at 296 K and they were
performed using pulsed field gradients along the z-axis.
Watergate two-dimensional N OESY experiments [ 38] at
296 K and at 286 K were registered at 800 MHz to identify
connectivities involving h istidines of the active site. In both
experiments 2048 (
1
H) · 1024 (
1
H) data points were
acquired with spectral w indows 9124 Hz (
1
H) · 9124 Hz
(
1
H); mixing times of 130 and 60 ms for t he experiments
acquired at 296 and 286 K, respectively, were used.
In order to detect the amide hydrogen–deuterium
exchange a series of
1
H-
15
N HSQC spectra on the sample
prepared dissolving the lyophilized protein in D
2
O solution
were collected at 800 MHz, at 296 K. 1024 (
1

H) · 256 (
15
N)
data points were acquired with spectral windows 12 019 Hz
(
1
H) · 4065 Hz (
15
N) for each spectrum. Each
1
H-
15
N
HSQC spectrum was acquired in 20 min every hour over a
24-h period. After 4 days from the dissolution in D
2
O one
experiment was a cquired to d etect the r emaining amide
protons.
Quadrature detection in the indirect dimensions was
performed and water suppression was achieved through the
WATERGATE sequence [39].
Data were processed with the standard Bruker software
packages (
XWINNMR
). Data analysis and assignment was
performed using the program
XEASY
(ETH, Zurich, Swit-
zerland) [40].

Structure calculation
NOE cross-peaks in three-dimensional
15
Nand
13
C-
NOESY-HSQC spectra and in two-dimensional N OESY
spectra were integrated and c onverted into upper distance
limits f or inte rproton distances with the program
CALIBA
[41]. The calibration curves for this conversion were adjusted
iteratively as t he structure c alculations proceeded. N OE
cross peaks due to couplings between the two subunits were
converted into upper distance limits using specific calibra-
tion curves. In the case of protons belonging to the ligand
histidines an independent calibration has been used for each
histidine. Stereospecific assignments of diastereotopic
1906 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002
protons have been obtained using the program
GLOMSA
[41]
and by the analysis of the HNHB experiment.
Backbone dihedral angle restraints / were derived f rom
3
J
HNHa
coupling constants by means of the appropriate
Karplus relationship. For
3
J

HNHa
values larger than 7 Hz
the / angle ranges between )155° and )80° while for values
lower than 4.5 Hz it ranges between )70° and )30° [36].
Backbone dihedral angle w for r esidue (i ) 1) was deter-
mined from the ratio of the intensity of the d
aN
(i ) 1,i)and
d
Na
(i,i) NOE found on the
15
N plane of residue (i)inthe
15
N-NOESY-HSQC. Ratio values of the residue (i ) 1)
larger than 1 are charact eristic of b sheets, with w values
ranging between 60° and 175°, while values smaller than 1
indicate a right handed a helix, with w values between )90°
and )10° [42]. v
1
torsion angle constraints were derived by
the intensity ratios between the volume integral, d
Nb
(i,i), in
the three-dimensional HNHB and the volume integral,
d
NH
(i,i), in the two-dimensional reference spectra, as
previously reported [37].
Structure calculations were performed using the program

DYANA
[43]. Fourteen-hundred random conformers were
annealed using the above constrains in 18 000 steps for the
initial calculations on a single subunit and in 22 000 steps
for the dimeric form. The dimeric nature of the protein was
taken into account by co nnecting the amino a cid sequence
of the two subunits with a chain of linkers composed of
atoms with a null Van d er Waals r adius. The metal ions
were included i n the calculations by adding special linkers
(pseudoresidues) in the amino-acid sequence following the
same procedure already used for the monomeric forms [26].
The linkers only define the metal–nitrogen distances, leaving
the conformation of the histidines completely free; the bond
angles at the copper and zinc ions are not imposed but can
freely change in the structural calculations, being only
determined by the e xperimental intrahistidine NOEs. The
presence of the disulfide bridge between Cys57 and Cys146
was checked through SDS/PAGE and through the analysis
of the
13
C shifts of the Cb of the cysteines. In the
calculations thr ee upper a nd three l ower distance limits
were used to enforce t he disulfide bond Cys 57–Cys146
[between the Sc moieties of the two Cys, 2.0 (lower) and 2.1
(upper) A
˚
, and between the Cb of one Cys with the S c of the
other, 3.0 (lower) and 3.1 (upper) A
˚
][44].

The program
CORMA
[45], which is based on relaxation
matrix calculations, was used to back calculate the NOESY
cross-peaks from the calculated structure to assess the
quality of the structure.
The final family was made up of 30 structures with the
lowest target function. Restrained energy minimization in
vacuum (REM calculations) was applied to each member of
the family using the program
AMBER
5.0 [46]. The s etup of
the program and the parameters for the metal ions are as
previously reported [21]. T he value of NOE and torsion
angle potentials h ave been applied with force constants of
50 kcalÆmol
)1
ÆA
˚
)2
(NOE), 32 kca lÆmol
)1
Ærad
)2
(/, Y)and
2kcalÆmol
)1
Ærad
)2
(v

1
).
The program
MOLMOL
[47] was used for identification of
hydrogen bonds (within the distance of 2.6 A
˚
between
donor and acceptor, t he N-H-O angle larger than 140° and
occurrence in at least 50% of the conformers).
The quality of the structure has been estimated by
Ramachandran plots obtained using the program
PROCHECK
-
NMR
[48].
RESULT AND DISCUSSION
Resonance assignment
Native dimeric SOD is co mposed of two identical subunits
which produce degenerate resonances. Although this was
already shown for the active site resonances from the
investigation on the Cu
2
Co
2
SOD derivative [ 49], it i s a
relevant result as X-ray data [32] often i ndicate different
conformations for the two subunits [10]. T hus, averaging
occurs on the NMR time scale, i.e. faster than seconds.
The proton a nd

15
N resonances are not well dispersed
and experience extensive overlap due to the specific folding
of the protein, characterized by extensive b sheet structure.
However, most of the
1
H-
15
N cross peak degeneracies
present in
15
N-HSQC spectrum were resolved in at least one
of the HNCA, HN(CO)CA, HNCO a nd HN(CA)CO-
TROSY-type spectra already performed for the backbone
assignment, reported by us [30].
The assignment of the resonances of the s ide chains was
performed through the analysis of three-dimensional
H(C)CH-TOCSY and (H)CCH-TOCSY spectra together
with
15
N-NOESY-HSQC and
13
C-NOESY-HSQC spectra.
In this way about 92% of the total proton resonances were
assigned. All the backbone proton and nitrogen resonances,
with the single exception of Phe64, were assigned. All the
nitrogen side chain resonances o f Asn a nd Gln, with the
exception of Gln153 (th e l ast o ne in each s ubunit), were
assigned. Ninety-nine percent of the backbone
13

Creso-
nances were assigned and about 86% of the
13
C side-chain
resonances. All the ring protons of the histidines of the
active site and o f His43 were ass igned through t he two-
dimensional NOESY map. The histidine coordination
mode was determined through
1
H-
15
N heteronuclear
experiments, by detecting the
2
J
15
N-
1
H coupling between
the imidazole nitrogen and nonexchangeable imidazole
protons.
Structure constraints and calculations
In three-dimensional
15
N- and
13
C-NOESY-HSQC spectra
and i n two-dimensional
15
N-NOESY s pectra, 356 6 NOE

cross peaks were assigned and converted into distance
constraints. Forty-nine dihedral / angles constraints were
obtained from the analysis of th e HNHA spectrum,
52 dihedral w angles were obtained from the
15
N-
NOESY-HSQC spectrum and 14 v
1
torsion angles from
the HNHB spectrum. A total of 45 proton pairs were
stereospecifically assigned with the program
GLOMSA
:16
protons belonging to bCH
2
,11toaCH, 1 to cCH
2
,1to
dCH
3
and 1 to cCH
3
;and15bCH
2
were assigned by the
analysis of the H NHB experiment. In each subunit the
metal ions were included by allowing copper t o bind to
Ne2 of His48 and His120 and to Nd1of His46, and zinc to
Nd1 of His63, His71, and His80 and to Od1 of Asp83.
Lower and upper distance limits of 1.8 and 2.3 A

˚
,
respectively, were imposed between t he metal i ons and
the donor atoms. Finally, 3276 upper distance limits were
generated of which 2853 were due to meaningful NOEs. All
the available information on the system, the linewidth and
the number of signals, lead to a dimeric species with
twofold s ymmetry. The NOEs a nd 115 dihedral angles
were initially used for the structural calculations of the
Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1907
monomeric species. During these initial s tructure calcula-
tions, the presence of a small but significant number of
NOEs (96) inconsistent with couplings with protons o f
residues of the same subunit, were identified and assigned
to connectivities between protons belonging to two differ-
ent subunits. They w ere introduced in the calculations at a
later stage after r efinement of the monomeric structure.
Only one NOE has a contribution from both inter and
intra subunit; no severe violation with respect to the
calibration was observed. For the calculations of the
dimeric s tructure, the intra s ubunit NOEs and dihedral
angle constrains were duplicated for each subunit and the
inter subunit NOEs were included. The number of
constraints, divided in classes, are listed in Table 1. The
number of experimental N OEs per residue per subunit is
reported i n Fig. 1.
From the final calculations, a family of 30 conformers,
with the l owest target f unction of 5.02 A
˚
2

(average value
5.99 A
˚
2
), was obtained with a n average violation p er residue
of 0.016 A
˚
. E ach c onformer o f t his f amily was refined
further through REM calculations. The rmsd to the mean
structure of the family is 0.79 ± 0.11 and 1.35 ± 0.09 A
˚
for the backbone and the heavy atoms, respectively (rmsd
calculated over the fragment 3–151 for the holo protein).
After the refinement, the rmsd values a re 0.78 ± 0.11 and
1.15 ± 0.09 A
˚
for t he backbo ne and the heavy atoms,
respectively. If the rmsd is evaluated for each subunit of the
protein the values drop to 0.65 ± 0.09 and 0.66 ± 0.10 A
˚
,
respectively, for the backbone of the two subunits. The
difference between rmsd of monomeric and dimeric species
is due to the indetermination of the reciprocal orientation of
the two subunits. The average total penalty for the REM
family of the dimeric p rotein is of 1.42 ± 0.07 A
˚
2
for the
distance constrains; while for the average structure the value

is 1.36 A
˚
2
. The rmsd values per residue, with the respect to
the average structure, are shown in Fig. 2.
General shape of the protein and comparison
with X-ray structures
A tube representation of the family of structures (back-
bone and metal ions only) is shown in Fig. 3. The family
of conformers was analyzed with
PROCHECK
-
NMR
and the
results of t he analysis are reported in Table 1. The
secondary structure elements are eight antiparallel
b strands and a short five-residue a helix, which, connected
by loop regions, produce the typical S OD Greek key fold.
The secondary structure part of the protein is well defined.
The average rmsd values for the segments involved in the
b barrel are 0.50 ± 0.08 A
˚
and 0.85 ± 0.06 A
˚
for the
backbone and all heavy atoms, respectively, which indi-
cates that the b strands are characterized by lower disorder
than the loops connecting them. If a s ingle subunit is
considered, the bbarrel rmsd values are 0.38 ± 0.06 A
˚

and 0 .7 7 ± 0.06 A
˚
, f or the backbone and a ll heavy
atoms, respectively. These values indicate that the
b strands in each subunit are well defined. The a helix
within the family of confor mers has an average rms d to
Table 1. Restraint violations and structural and energetic statistics for the solution structure of reduced human SOD.
RSM violations per experimental distance constraint (A
˚
)
b
REM
a
(30 structures) <REM>
a
(mean)
Intraresidue (723) 0.0251 ± 0.0013 0.0245
Sequential (1546) 0.0124 ± 0.0010 0.0119
Medium range (924)
c
0.0149 ± 0.0009 0.0140
Long range (2513) 0.0115 ± 0.0005 0.0109
Total (5706) 0.0147 ± 0.0004 0.0147
RSM violations per experimental dihedral angle constraints (deg)
b
Phi (98) 1.55 ± 0.21 1.54
Psi (104) 0.52 ± 0.24 0.0
Chi1 (14) 0.41 ± 0.26 0.0
Average number of violations per structure lower than 0.3 A
˚

Intraresidue 51.4 ± 3.8 51
Sequential 42.7 ± 4.5 41
Medium range 31.9 ± 3.3 26
Long range 52.8 ± 3.7 53
Total 178.7 ± 6.3 171
Phi 8.1 ± 1.9 8
Psi 1.5 ± 0.9 0
Chi1 1.3 ± 0.6 0
Average no. of NOE violations larger than 0.3 A
˚
00
Structural analysis
d
% of residues in most favourable regions 71.6 73.6
% of residues in allowed regions 25.6 24.4
% of residues in generously allowed regions 2.3 2.1
% of residues in disallowed regions 0.6 0
a
REM indicates the energy minimized family of 30 structures, <REM> is the energy minimized mean structure obtained from the
coordinates of the individual REM structures.
b
The number of experimental constraints for each class is reported in parentheses.
c
Medium
range distance constraints are those between residues (i,i +2)(i,i +3)(i,i + 4) and (i,i + 5).
d
As it results from the Ramachandran plot
analysis.
1908 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the mean structure of 0.39 ± 0.01 A

˚
and 0.91 ± 0.21 A
˚
,
for the backbone and all heavy atoms, respectively. These
values drop to 0.16 ± 0.08 A
˚
and 0.65 ± 0. 26 A
˚
when a
single subunit is c onsidered. The c omparison of the
present structure with the X -ray structures of the human
oxidized protein ( 1SOS) [11] and its G 37R mutant [50]
show that the protein has the same folding in solution and
in solid state.
The loops connecting the secondary s tructure elements
can be divided in two groups: the loops I, III and V are quite
well defined, while loops II, IV and VI are more disordered.
The odd loops are located on the opposite side of the barrel
with respect to region involved in the subunit–subunit
interface. The even loops are in part located at the subunit–
subunit interaction. The first part of loop IV (49–62) shows
(Fig. 4 ) a much lower backbone rmsd in the present
Fig. 1. Number of intraresidue (white), sequential (light grey), medium-range (grey) and long-range (black) intra subunit NOEs per residue (bottom)
and number of inter subunit NOEs per residue (top) in human reduced native SOD.
Fig. 2. Average rmsd values of backbone (j) and heavy atom (h) on two subunits per residue with respect to the average structure of human reduced
native SOD (bottom). Backbone (j) and heavy atom (h) rmsd values of a single subunit per residue with respect to the average structure of human
reduced native SOD (top).
Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1909
structure than in t he monomeric Q133M2SOD structure.

This can be related to the occurrence of interactions with the
other s ubunit that m inimize the exposure to solvent of
residues at the interface and stabilizes a single conformation
for them. Indeed, in the segment 50–59 of Q133M2SOD,
five backbone HN signals were not assigned, probably due
to line-broadening as a consequence of p roton exchange
with solvent due to their surface location. This is consistent
with the analysis o f t he amide h ydrogen–deuterium e x-
change behaviour previously reported [51]. The other loops
are still disordered even in the dimeric form. Therefore, this
behaviour suggests that this is a feature typica l of this part o f
the protein independent of its quaternary structure. The
change in conformation of loop 50–59 upon dimerization is
reflected also on the location of Cys57, which can or cannot
perform a H-bond with the side chain of Arg143 depending
on its conformation (see below).
FALS mutations are spread over the entire molecule but
a higher density of mutations are clustered in a few regions
of the protein: at the interface between the two subunits
(mainly in loop IV and b 8), in the odd loops and at the
corresponding end of the b barrel, and in the even loops
[52–56]. Some of the residues involved in FALS m utations
are conserved i n SOD structures from different species
(Fig. 5) [11]. The FALS mutations located in the first region
are thought [11] to significantly destabiliz e the subunit–
subunit contacts. This is in agreement with the NMR d ata
Fig. 5. Close-up of one subunit of human reduced SOD showing FALS
mutations. The F ALS mutations located in odd loo ps are shown in
gray, those in b strands are in black and located in the region close to
the subunit–subunit interaction are coloured black and the residue

labels are underlined.
Fig. 3. Tube representation of the family of 30 structures of human reduced native SOD obtained with
DYANA
calculations and refined with
REM
calculations. Elements of secondary structure are highlighted (gray, b structure; black, a struc ture). The drawing has been produced with
MOLMOL
[47].
Fig. 4. Comparison o f rmsd values to the average structure for the
backbone between dimeric SOD (s) and E133QM2SOD (.).
1910 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002
on the solution structures [27,57] and on mobility studies
[30] of monomeric variants and human dimeric SOD, where
it has been shown that the absence of interactions with the
other subunit has sizable effects on enzymatic stability and
activity.
Hydrogen–deuterium exchange
A total of 104 amide protons out of 147 were still present in
the
1
H-
15
N HSQC spectrum acquired 6 h after the disso-
lution of the lyophilized sample in D
2
O. Fifty-one residues
are located in regions having a defined secondary structure
as they are involved in an extensive H-bond networks which
stabilize the b barrel structure typical of this protein. Few
exceptions are observed in one of the b sheets (b6) where

amide protons belonging to three residues (Asp96, Ser98,
Glu100) out of six, exchange within 40 min and those
belonging to Asp101 within 1 h 40 min. Also the a helix
shows exchanging amide protons in the time range between
40minto12h.
After 4 days 85 peaks, mostly belonging to the b barrel
and to loops III and VII were still present.
Metal sites
In Fig. 6 the active site is shown and compared with that of
oxidized human SOD. All the metal ligands are well defined
in a single conformation. For all the ligands, the rmsd value
calculated for all heavy atoms is smaller than 0.8 A
˚
,avalue
that is similar t o t hat obtained for secondary structure
elements. The ligand conformation is also very close to that
observed in all the structures available for eukaryotic SOD,
either based on X-ray or NMR analysis in solution, dimeric
or monomeric. The only exceptions are His63 and t he
copper ion. His63 experiences a l arger variability among
the various structures and its orientation i s dependent on the
copper oxidation state. I n oxidized SOD, His63 is coordi-
nated to the Cu ion through its Ne2, the distance between
copper and Ne2 being about 2.1 A
˚
in the human structures
(1SOS) [11] and about 2.7 A
˚
in a mutant (G37R) [50]. In the
reduced state, the bond between Cu and His63 is broken,

producing an increase in distance between the two. In the
case of the reduced dimeric yeast enzyme this distance
increases to 3.2 A
˚
[20]. In the present structure the reduced
copper is clearly tricoordinated, as expected from the data
on the monomer. Indeed, upon copper reduction His63
becomes p rotonated at t he Ne2 position, the bound
proton resonating at 12.3 p.p.m. and the distance
between copper and Ne2being3.3A
˚
. In the present
structure the major structural changes induced by copper
reduction is the movement of the copper ion which moves
away from His63, experiencing a displacement of about
1.7 A
˚
with respect to the oxidized enzyme. So, the increased
Cu–His63 distance, in the reduced state, is due to a
movement of copper more than to a change in conforma-
tion of His63. The position of the other metal ligands, in the
present structure, is very close to that found in the reduced
dimeric yeast isoenzyme [20], whereas the copper ion
positions in the two structures differ by about 1.0 A
˚
.It
should be noted, however, that in the reduced yeast
structure Cu a nd Ne2 of His63 are at a distance shorter
than the sum of their van der Waals radii.
The Zn ion does not experience significant movement

from its site compared to the other structures. In reduced
monomeric human mutants (Q133M2SOD and M4SOD)
the Zn ion moves farther from the copper ion. The distance
between the metals in the present structure is 7 A
˚
,thisis
similar to that in the dimeric yeast isoenzyme (6.7 A
˚
), while
in the human oxidized structure it ranges between 6.1 A
˚
to
6.3 A
˚
.
About the active site channel
The active site channel is located between the electrostatic
loop VII (120–144), implicated in assisting and increasing
the affinity for the active site of substrate, and loop IV (49–
82). A network o f H-bonds between the s ide chains o f
some residues belonging to loop VII plays a crucial role in
increasing the diffusion rates of the superoxide radical
inside the cavity [33]. Comparing X-ray structures (1SOS,
G37R and 1JCV) with the present one, it can be observed
that the o rientation of the a helix is the s ame i n a ll the
structures and the backbone remains almost unaltered. In
contrast the side chains experience different conformations:
Glu132 shows a different orientation in each of the
structures and in each of the subunits in the crystal cells,
whereas n o meaningful comparison can be carried out for

Glu133, which shows disorder in the side chain. Ser134
and T hr135 are quite ordered in the present structure, but
they have a different orientation of t he hydroxyl group
with respect to the X-ray structures. Side chain of Lys136
has different orientations in each of the X-ray structures.
The present one is closer to that in 1SOS and G 37R
structures. Thr137 shows no significant changes in the
orientation of th e side chain although a movement towards
Arg143 is observed, which slightly decreases the width of
theactivesitechannel.
Thr58 and Glu133, with Glu132, define the opening of
the active cavity. The width is about 13 A
˚
(distance between
Thr58 Cc and Glu133 Oe), which is d ecreased by % 1A
˚
with respect to 1SOS and % 2A
˚
with respect to G37R.
Arg143 with Thr137 form a ÔbottleneckÕ for the active site,
which excludes sterically large nonphysiological anions. In
Fig. 6. Active site of the family conformers of the reduced human dimer
(blue) and of the oxidized human dimer (red).
Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1911
the present structure also these residues are slightly closer
than the X-ray struct ure.
Arg143 i s important in orienting the superoxide anion
towards the Cu ion. Comparing the present structure with
1SOS, G37R and 1JCW, the side chain of Arg143, in most
of the conformers shows no significant changes in the

orientation, while in the case of the monomers
(Q133M2SOD and M4SOD) the Arg143 side chain has a
different orientation (Fig. 7). Cys57, with residues 58 and
61, was proposed to stabilize the orientation of Arg143
[26,27], as a result of hydrogen bonds between the side chain
of Arg143 (protons of Ng1andNg2 groups) and backbone
carbonyls of Cys57, Thr58 and Gly61. The H-bonds
involving C ys57, Thr 58 a nd Gly61 a re present in several
conformers. The latter three residues are defined by 23, 24,
20 NOEs, respectively. Furthermore, Cys57 forms a disul-
fide bond with Cys146, which is defined by 36 NOEs. Side
chains of Arg143 and Cys57 are defined by 26 and seven
NOEs, respectively. The Cu–Ng1andCu–Ng2 average
distances of Arg143 are 7.2 and 7.3 A
˚
from copper, while in
the oxidized human protein the distances are 5.8 and 7.0 A
˚
.
This is consistent with the already discussed movement of
the copper ion upon reduction.
Cys57 seems to play a fundamental role in the process of
copper transfer from the copper chaperone for SOD (CCS)
and SOD itself as shown by t he recently solved structure of
the CSS–SOD complex [58]. In the latter structure, Cys229
of CCS forms a disulfide bond with Cys57 o f SOD [58],
which t herefore is not interacting any longer with Arg143.
The guanidinium group of the latter residue in the complex
is very far away from the site where copper should b e
introduced and is pointing t owards the c haperone. The

conformation of Arg143 is extremely sensitive to the
position of Cys57 [26,27]. In the monomeric species, where
Cys57 experiences conformational equilibria, still maintain-
ing the disulfide bond, Arg143 is further from c opper than
in the wild-type protein but closer than in the copper-free
SOD in the complex. In the present solution structure of
wild-type SOD where the Cys57 is quite rigid, Arg143
assumes the optimal conformation respect to the c opper.
Therefore it seems that Arg143 is experiencing a movement
that leads it to assume the correct conformation when SOD
is passing from the complex with CCS (where SOD is in a
monomeric state) to the single monomeric protein, to the
final dimeric structure.
Relevant H-bonds
A network of H-bonds in dimeric human oxidized SOD [8]
was proposed to play an important role in building the
Greek key structure and in designing the metal binding site
and the active cavity of the system. The analysis of the
H-bonds in the present structure has been carried out with
MOLMOL
program [ 47] on the final structure. Except a few
cases d iscussed later the hydrogens involved in H-bonds do
not exchange in D
2
O. Some of the H-bonds present in the
human oxidized X-ray structures are observed also in the
solution structure. The H-bonds among ring hydrogens of
His43 and backbone carbonyls of Thr39 and the Cu-ligand
His120 ar e present in almost all the conformers of the
family. Ho wever the He2 of His43 do exchange in D

2
O
indicating solvent exposure of such H-bond. H-bonds
involving ring hydrogens of His43 are important in linking
the loop III to the b barrel and the active site. The presence
of these H -bonds is co nsistent with th e N MR observation of
two HN ring protons signals for His43 (which is not
involved in metal binding) at pH 5.0. In the present
structure, the side c hain Od of Asp124 forms a H -bond
with the ring hydrogens He2 of His71 and of His46. Asp124
constitutes a long-range bridge between the copper site and
the zinc site [8]. Mutations of residue 124, which have been
found in FALS proteins, affect mainly the zinc site and its
affinity for the zinc ion [59]. These mutations might produce
zinc deficient species that h ave b een s hown to gain
peroxynitrite producing a ctivity, a possible c ause of the
FALS diseas e [ 60–63]. A conserved H -bond between
backbone HN of His71 and CO of Thr135, important in
stabilizing the active site channel, is present in several
conformers. Thr135 belongs to the six residue helix involved
in the recognition and in the electrostatic guidance of the
superoxide anion. The amino acid site chains of Glu132,
Glu133, Lys136 and Thr137 are involved in a hydrogen
bonding network [33]. In the present structure this H-bond
network is maintained.
For the FALS mutations located in the region constituted
by odd loops and one end of b barrel (Fig . 5), as for
example G37R, the absence of some H-bonds in the b
hairpin region (loop V) is supposed to be responsible of the
misfunction of the enzyme [50]. In the present structure all

the odd loops are well defined and this is consistent with the
presence of a network of H-bonds that stabilizes this part of
theprotein.ThisregioniscenteredonLeu38,calledthe
ÔplugÕ of one end of the bbarrel [ 11], which fills a cavity
formed by an array of apolar aminoacids present in
different b strands (Ile35, the ring face of His43 and
Leu144) and loop I (Val14). T hus producing a packed
arrangement, crucial for correct enzymatic f unction and
protein stability [64]. Conserved H-bonds observed in this
crucial part of the protein, observed in the present structure
and identified with the program
MOLMOL
, are summarized
in Table 2. The H-bond connecting loop III and loop V,
containing b hairpin (HN of Leu38 and CO of Gly93) and
the H-bond between HN of Gly93 and CO of Asp90 (loop
Fig. 7. View of the active channel of human reduced native SOD. Ori-
entation of the s ide chain of Arg143 is reported for t he re duced human
dimer (blue), for the oxidized h um an dimer (red), for t he Q133M2SOD
(cyan) a nd for the reduced e nzyme derived from yeast (yellow). The Cu
ion is shown as a sphere and the a helix is in orange.
1912 L. Banci et al. (Eur. J. Biochem. 269) Ó FEBS 2002
V) and between HN of Asp92 a nd side chain carboxylic
group of Asp90 are well conserved in all conformers of th e
present family and in the X-ray structures (1SOS) even if
some difference in the stability of H-bond could be present.
Indeed the amide proton of Asp92 disappears in D
2
Oafter
about 2 h, whereas the others a re still present four days after

the dissolution in D
2
O. In the F ALS muta nt G37R t he
H-bond between HN of Asp92 and the carboxylic group of
Asp90 is p resent in only one of the two subunits [50]. The
loss of this hydrogen bond in the G37R mutant [50] was
proposed to allow a n increase fl exibility in t he b hairpin,
with respect to the wild-type protein; the latter, in fact, is
characterized by the absence of motions in the ps-ns
timescale in this region [30]. Gly41, Gly37 and Gly93 seem
necessary to support main chain conformations and the
packing interaction in the h ydrophobic plug [64]. Gly41 is
involved in H-bond with Ala89 that, in its t urn, is close t o
the b-hairpin which i s further stabilized by the H -bond
between Asp90 and Val94. The presence of extensive
H-bond networks seem to play a fundamental role in
stabilizing the secondary and tertiary structure of the
protein.
CONCLUSIONS
The solution structure of dimeric human reduced Cu
2
Zn
2
SOD was determined to a satisfactory degree of resolution.
The two monomers are identical on the NMR t ime scale.
The elements of secondary structure are the same as in the
X-ray structures and well resolved as well as the three odd
loops and t he first part of loop IV, at t he inter–subunit
interface. The even l oops, have a r elatively high r msd. A
similar behavior i s observed in a recently reported X-ray

structure of bovine SOD [32]. The structure i s also similar t o
the solution structure of the monomeric mutants with the
exception of the s ignificantly better definition of the first
part (49–63) of loop IV, which is disordered in the
monomers and experiences significant local mobility. T he
active channel is formed by the electrostatic loop VII, where
charged residues important in catalysis lie, and loop IV,
where Cys57 is located. Upon dimerization, loop IV looses
the conformational exchange equilibria, occurring in the
ms-ls time range, and assumes a conformation which f avors
the formation of the hydrogen bond network. The optimal
conformation of the side chain of Arg143 is ensured by the
formation of H-bonds between its terminal guanidinium
group and the backbone oxygen atoms of Cys57 and Gly61
(loop IV). The latter network contributes to determine the
optimal orientation o f the strategic residue Arg143 and
reduces its mobility i n the subnanosecond time sc ale. In
contrast, the increased mobility, in the subnanosecond time
scale, of the electrostatic loop VII (a helix) could assist O
2
–
in sliding inside t he active cavity, w here it reaches the correct
position helped b y t he interaction with the correctly oriented
Arg143. The optimal orientation of Arg143 is found also in
a wild-type bacterial SOD [65], which i s naturally mono-
meric and where Cys57 (human numeration) is still
H-bonded to Arg143.
The copper site in the present d imeric structure i s in a
position similar to that of the monomeric mutants. Because
X-rays, when irradiating the crystals, may change the

oxidation state or the s olid state a nd may i nduce subtle
structural changes, the present characterization of the
reduced active site represents a f urther reliable p icture of
the reduced protein. Upon reduction, copper moves inside
the active cavity. This is consistent with the earlier proposal
[20,28,66] that the superoxide ion hardly reaches copper (I)
but rather interacts with the e2protonofHis63andis
activated by this interaction for t he transfer of one electron
from copper (I). Finally, the strong H-bond network
involving odd loops and one end of the b barrel (Table 2)
is observed in solution. It may be relevant that some FALS
mutants disrupt this network, giving them the capability of
catalyzing other toxic reactions.
In conclusion, the present structure of the dimeric wild-
type SOD, although at lower resolution with respect to the
X-ray structures, provides a clear refined picture of the
relevant residues i n solution a nd allows a thorough under-
standing of the effects of establishing a quaternary structure.
ACKNOWLEDGEMENTS
This work was supported by the European Com munity ( Contract
number HPRI-CT-1999-00009 and QLG2-CT-1999-01003), by Italian
CNR (Progetto Finalizzato Biotecnologie 99.00286.PF49 and
99.00950.CT03) and by MIUR-ex 40%.
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SUPPLEMENTARY MATERIAL
The f ollowing material is available f rom h ttp://www.
blackwell-science.com/products/jo urnals/su ppmat/EJB/
EJB2840/EJB2840sm.htm
Table S1. Resonance assignments.
Table S2. Experimental NOE intensities of reduced human
SOD.
Table S3. v
1
torsion angle constraints for a single subunit in
reduced human SOD.
Table S4. / torsion angle constraints f or a single subunit in
reduced human SOD.
Table S5. w torsion angle constraints for a single subunit in
reduced human SOD.
Table S6. Stereospecific assignments diastereotopic pairs for
a single subunit in reduced human SOD.
Ó FEBS 2002 Solution structure of dimeric Cu,Zn SOD (Eur. J. Biochem. 269) 1915