Báo cáo khoa học: The crystal structure of annexin Gh1 from Gossypium hirsutum reveals an unusual S3 cluster Implications for cellulose synthase complex formation and oxidative stress response potx

The crystal structure of annexin Gh1 from
Gossypium hirsutum
reveals an unusual S
3
cluster
Implications for cellulose synthase complex formation and oxidative stress response
Andreas Hofmann
1
, Deborah P. Delmer
2
and Alexander Wlodawer
3
1
Institute of Cell & Molecular Biology, The University of Edinburgh, Edinburgh, Scotland;
2
The Rockefeller Foundation,
New York, USA;
3
Macromolecular Crystallography Laboratory, NCI at Frederick, Frederick, Maryland, USA
The three-dimensional crystal structure of recombinant
annexin Gh1 from Gossypium hirsutum (cotton fibre) has
been determined and refined t o t he final R-factor of 0.219 at
the resolution of 2.1 A
˚
. This plant annexin consists of the
typical Ôannexin foldÕ and is similar to the previously solved
bell pepper annexin Anx24(Ca32), but significant differences
are seen when compared to the structure of nonplant
annexins. A comparison with the structure of the mamma-
lian annexin AnxA5 indicates that canonical calcium bind-
ing is geometrically possible within the membrane loops in

domains I a nd I I of Anx(Gh1) in their present co nformation.
All plant annexins possess a c onserved tryptophan residue in
the AB loop of the first domain; this residue was found to
adopt both a loop-in and a loop-out conformation in the bell
pepper annexin Anx24(Ca32). In Anx(Gh1), the conserved
tryptophan residue is in a surface-exposed position, half way
between both conformations observed in Anx24(Ca32). The
present structure reveals an unusual sulfur cluster formed
by two cysteines and a methionine in domains II and III,
respectively. While both cysteines adopt the reduced thiolate
forms and are separated by a distance of about 5.5 A
˚
,the
sulfur atom of the methionine residue is in their close vicinity
and apparently interacts with both cysteine sulfur atoms.
While the cysteine residues are conserved in at l east five p lant
annexins and in several mammalian members of the annexin
family of proteins, the methionine residue is conserved only
in three plant proteins. S everal of these a nnexins carrying t he
conserved residues have been implicated in oxidative stress
response. We therefore hypothesize that the cysteine motif
found in the present structure, or possibly even the entire
sulfur cluster, forms the molecular basis for annexin function
in oxidative stress response.
Keywords: calcium; cellulose synthase; cotton; oxidative
stress response; plant annexin.
Oxidative stress is a health-threatening phenomenon in
many biological systems that results from the effects of
partly reduced oxygen species, such as superoxide radical
(O

ÁÀ
2
), hydroxyl radical (OHÆ), and hydrogen peroxide
(H
2
O
2
). These s pecies are by-products of normal aerobic
metabolism and result from successive single electron
transfers from/to oxygen . Partially reduced oxygen species
are involved in DNA damage, lipid peroxidation, and
protein denaturation. Through apoptosis and necrosis,
these types of cellular damage can give rise to several
pathological symptoms observed in diseases such as cancer,
arthritis, and muscular dystrophy, as well as to genetic and
nervous disorders [1–4].
Mammalian annexins A1 [5], A5, and A 6 [6], as well as
plant annexins from Medicago sativa [7] and Arabidop-
sis thaliana [8,9], have been implicated in oxidative stress
response. In particular, it has been shown that an annexin-
like protein from Arabidopsis, Oxy5, is able to rescue
Escherichia coli DoxyR mutants from H
2
O
2
stress . Cot ton
fibre annexins have been shown to colocalize with cellulose
synthase and to have an inhibitory effect on glucan synthesis
[10]. In a recent study [11] a redox-dependent model for
cellulose synthase complex formation was proposed, which

also implicates the cotton annexins in putative red ox
activities.
While structural biology of vertebrate annexins is well
established and has yielded a wealth of information about
these proteins, their plant relatives are less well character-
ized, although known for 13 years [12]. As detailed in a
continuously updated list [13], annexins have been found in
every plant where a search was initiated. Examples include
Anemia phyllitidis (fern), Anemia thaliana (mouse-ear cress),
Capsicum annuum (bell pepper), Dryopteris filix-mas (fern),
Gossypium hirsutum (cotton), Lavatera thuringiaca (mal-
low), Lycopersicon esculentum (tomato), M. sativa (alfalfa),
Nicotiana tabacum (tobacco), Pisum sativum (pea), Solanum
tuberosum (potato), and Zea mays (maize). Two d istinct
plant annexins occur m ost frequently and s how very high
sequence similarity throughout different plants. Despite
having similar molecular weights, both proteins migrate
differently on SDS/PAGE and the apparent molecular mass
has thus been added to their annotation until a final
classification into the new annexin nomenclature is done.
Based on these observations, the idea of two distinct
Correspondence to A. Hofmann, Institute of Cell & Molecular Biology,
The University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.
Fax: + 44 131 6508650, Tel.: +44 131 6505365,
E-mail:
(Received 7 February 2003, revised 20 March 2003,
accepted 8 April 2003)
Eur. J. Biochem. 270, 2557–2564 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03612.x
annexin subfamilies in plants (Sp32, Sp38) was put forward
[14]; however, the recent report of a total of seven annexin

homologues in Arabidopsis [15] raises the question whether
annexins in plants might also appear as a diverse multigene
family, in common with their mammalian relatives.
Calcium binding has been identified as a landmark
feature of animal, plant, and metazoan annexin proteins. As
structurally established for annexin A5 [16], the canonical
type II calcium binding sites are found within the AB loops
of each domain and are provided by the endonexin sequence
K-G-X-G-T-{38}-D/E [17]. Typically, the coordination
sphere around the cation is a pentagonal bipyramid with
a backbone carbonyl group and a water molecule in apical
positions. Another water molecu le, three backbone carbo-
nyl groups, and the acidic residue from the conserved motif
form the base of the bipyramid. Because only one side chain
is involved in creating this site, there is no stringent apriori
requirement that the side chains within the endonexin
sequence be conserved. A d ifferent amino acid sequence
with a suitable loop conformation might act as proper
calcium binding site as well.
Type III and AB¢ sites, in contrast, are constituted by
one or two backbone carbonyl groups and a neighbouring
bidentate acidic residue and coordinate the calcium ion
together with several water molecules. Type III binding sites
are the only ones observed with DE loops. It has been
concluded that calcium bound in the AB loops is responsible
for membrane a dsorption, while the calcium harboured in
DE sites increases the binding affin ity in general [18].
While the primary structure of plant annexins reflects the
characteristic fourfold repeat, there is variation in the loops
harbouring the endonexin sequence. The motif is conserved

only in the first domain, occurs with quite some variations in
the fourth domain, and is not present i n the second and
third domain.
The first three-dimensional structure of a plant annexin,
Anx24(Ca32), proved that the plant proteins do indeed
possess the same characteristic annexin fold that has been
found in their mammalian and metazoan relatives [19].
Structurally, the most striking difference between vertebrate
and plant annexins is the convex (membrane-binding) side.
In the case of Anx24(Ca32), a number of hydrophobic and
aromatic residues are found on the surface of the mem-
brane-binding side. When comparing the membrane-bind-
ing loops of Anx24(Ca32) and AnxA5, it becomes clear that
the plant protein is not able to bind metal cations in the
conformation found in the crystal structure. Neither the first
nor the other domains are able to coordinate cations in these
regions, as positively charged residues in the close vicinity
present a repulsive force.
While various biochemical reports provide evidence that
plant annexins do bind calcium ions, it i s not clear so far
how and where the cations are accommodated in the
protein. Obtaining a crystal structure of the calcium-bound
form of Anx24(Ca32) proved extremely difficult, since the
protein was hard to crystallize and did not bind calcium
either by cocrystallization or soak methods. In order to try
another s ystem for these studies, we e mployed a different
plant protein, Anx(Gh1) from cotton, as a ÔprototypeÕ plant
annexin. Anx(Gh1) shares 72% identity with Anx24(Ca32)
and probably belongs to the class of Sp32 annexins.
In the current study, we purified and crystallized

recombinant Anx(Gh1) a nd determined its three-dimen-
sional crystal structure in the calcium-free form. A
comparison of the membrane binding loops with AnxA5
and Anx24(Ca32) reveals that canonical calcium binding
in the loops of domain I might be possible in Anx(Gh1),
in contrast to the bell pepper annexin. The protein
contains a highly unusual sulfur cluster formed by two
adjacent cysteine residues i n their reduced forms and a
methionine residue. The cluster is likely to be involved in
redox reactions and might constitute the m olecular basis
of oxidative stress response by annexins.
Materials and methods
Purification of recombinant protein
Cloning and construction of an N-terminal His
4
-fusion
protein has been described earlier [20]. T he recombinant
protein of Anx(Gh1) carried a hexapeptide extension
MAHHHH and was expressed in Escherichia coli
BL21(DE3) cells. A total of 8 L of LB medium (50 mgÆL
)1
ampicillin) were inoculated with an overnight culture of 1 L.
The cells were grown a t 37 °C until the absorbance at
600 nm exceeded 1.0. Induction was carried out with
0.5 m
M
isopropyl t hio-b-
D
-galactoside; at that t ime, the
concentration of ampicillin was increased twofold. Cell

growth was continued for 4–6 h.
The cells were harvested and lysis was performed by two
cycles of a freeze-thaw protocol. Cell debris was separated
by centrifugation for 30 min at 100 000 g. The supernatant
was applied to a Ni
2+
-nitrilotriacetic acid column equili-
brated with 100 m
M
NaCl, 20 m
M
Tris (pH 8.0). After
extensive washing of the column, a stepwise elution protocol
was performed with 20 m
M
,50m
M
, 100 m
M
, and 200 m
M
imidazole in equilibration buffer. The protein eluted at
50–100 m
M
imidazole and appropriate fractions were
pooled. In a second step, the recombinant protein was puri-
fied by anion exchange chromatography with Q-sepharose.
Pooled fractions obtained by affinity chromatography were
diluted threefold with 20 m
M

Hepes (pH 8.0) and applied to
a Q-Sepharose column. After a short washing, the protein
was eluted with a linear gradient 0–1
M
NaCl in 20 m
M
Hepes (pH 8.0). Anx(Gh1) eluted at 230–350 m
M
NaCl.
Concentration was carried out by ultracentrifugation using
Millipore Centricon devices.
Crystallization
Crystals of recombinant Anx(Gh1) were obtained using the
hanging-drop vapour-diffusion method. Droplets consisted
of 3 lLproteinand3lL reservoir solution equilibrated
against 300 lL reservoir solution at 285 K. The crystals
grew in about 8 weeks from 1.7
M
(NH
4
)
2
SO
4
,0.1
M
Hepes
(pH 7.0). Several crystals obtained from similar conditions
(pH 6.0–7.0) were soaked in mother liquor in the presence
of 2–15 m

M
CaCl
2
for b etween 2 h and 3 day, in an attempt
to obtain crystals of a calcium-bound form of Anx(Gh1).
Also, cocrystallization was attempted with calcium concen-
trations of 2–15 m
M
in the presence and the absence of
1.4 m
M
phosphatidylcholine.
2558 A. Hofmann et al. (Eur. J. Biochem. 270) Ó FEBS 2003
X-Ray data collection and structure solution
While diffraction obtained from crystals with the in-house
equipment was limited to 3.2 A
˚
, the maximal resolution
achieved at the synchrotron beamline X9B (NSLS, Brook-
haven National Laboratory) was 2.1 A
˚
. The structure
was solved using the synchrotron data set GH1–3A4, which
was collected at a wavelength k ¼ 0.97950 A
˚
.Datawere
collected from one crystal in two runs in order to minimize
spot overlap due to the considerable length of the z-axis of
the unit cell. In the first run, reflections between 40 and 3.2 A
˚

were recorded, while in the second run the detector was
moved closer to the crystal to record reflections between 8
and 2.1 A
˚
.Dataprocessingwascarriedoutwiththe
programs
DENZO
and
SCALEPACK
[21] and the data collec-
tion statistics are summarized in Table 1.
The diffraction pattern indicated a trigonal space group
with approximate cell dimensions of a ¼ b ¼ 61 A
˚
and c ¼
215 A
˚
. Two-fold axes were detected parallel t o [210] and
[120] in the self-rotation function calculated with GLRF
[22]. This rendered P3
1
12 and P 3
2
12 as the possible space
groups.
The structure was solved by molecular replacement
with AMoRe [23] starting from a poly Ala model of
Anx24(Ca32) that excluded the IAB loop region. A unique
solution was found in the space group P3
1

12 (correlation
coefficient: 0.76; next peak at 0.63), yielding an R-factor of
0.381. The asymmetric unit contains one molecule as
already indicated by the Matthews coeffi cient [24] of 3.2,
corresponding to 62% water content.
Structures of putative complexes of the protein obtained
by cocrystallization or soaking were later determined
by molecular replacement with the newly determined
Anx(Gh1) structure (see below) as the search model. Native
and anomalous difference fourier maps [25] were inspected
for the presence of calcium; anomalous maps never
contained peaks higher than 5.4 r, indicating that calcium
has not been successfully bound.
Model building and refinement
The poly-Ala m odel o f molecule 1 of Anx24(Ca32) as
positioned by AMoRe was subjected to rigid body
refinement with the four domains constituting four
independent rigid bodies. Crystallographic refinement
calculations were performed with
CNS
v. 1.0 [26] employ-
ing the conjugate gradient method and a maximum
likelihood target function. The initial model was rebuilt
with the program O [27] and subjected to extensive cycles
of computational refinement interspersed with visual
inspection and manual fitting. Subsequently, the alanine
residues were replaced by the proper side chains. The
revised amino acid sequence of Anx(Gh1) [20] was
unambiguously confirmed by the e lectron density. Typical
protocols consisted of a positional refinement followed by

simulated annealing, grouped and individual B-factor
refinement, and the final positional refinement. A flat
bulk-solvent model and overall anisotropic B-factor
correction were applied t hroughout the procedure. The
structure was refin ed to the final R-factor of 0.219 ( R
free
¼
0.280) with reasonable overall geometry, as monitored
with the program
PROCHECK
[28]. The refinement statistics
are summarized in Table 1. Coordinates and structure
factors have been deposited with the PDB under accession
number 1N00.
Figure preparation
Figures were prepared with
MOLSCRIPT
/
BOBSCRIPT
[29,30] using the
JAVA
application
BLUESCRIPT
for gener-
ating input scripts (A. Hofmann, unpublished data). The
objects created in such a manner were rendered with
POVRAY
[31].
Results and discussion
Crystallization

The main problem with structural studies of plant annexins
is the difficulty of obtaining crystals, since precipitation is
the predominantly observed behaviour. For this reason, we
were searching for a plant annexin which would crystallize
more readily than the previously reported annexin from bell
pepper [19]. A detailed elucidation of the o ligomerization
behaviour of plant annexins yielded calcium-independent
monomer-trimer e qu ilibria f or annexins 23(Ca38),
24(Ca32), and Gh1, whereas Anx(Gh2) exists in a monomer-
dimer e quilibrium [20]. Elution profiles from gel filtration
identified Anx(Gh1) as the annexin with the highest
monomer content in this series and, coincidently, it is this
protein which can be crystallized much more successfully
than the other ones.
Table 1. Data collection and refinement statistics. Values for the last
resolution shell (2.23–2.10 A
˚
) are given in parentheses.
Data set GH1–3A4
Data collection
Space group P3
1
12
Cell dimensions (A
˚
) 61.05, 61.05, 215.36
Resolution (A
˚
) 40–2.1
Number of measurements 808320

Number of independent reflections 27412
Completeness 100% (100%)
Multiplicity
a
7
R
merge
0.043 (0.433)
Refinement
No of reflections in working set/test set 24054 (3750)/
2656 (419)
Visible residues 4–321
Number of non-H atoms 2549
Solvent statistics: number of water
molecules/sulfate ions
181/3
R/R
free
b
0.219 (0.300)/
0.280 (0.377)
Average B-factor for all atoms (A
˚
2
) 51.9
Ramachandran plot: Residues
in most favoured/additionally
allowed/generously allowed region (%)
87.5/11.5/1.0
a

Estimated from
SCALEPACK
output;
b
R
free
defined in [35].
Ó FEBS 2003 X-ray structure of Anx(Gh1) (Eur. J. Biochem. 270) 2559
Structure of Anx(Gh1) in comparison with Anx24(Ca32)
and AnxA5
The three-dimensional crystal structure of annexin G h1
contains the typical annexin fold, well known from the
studies of other members of this protein family (see
Fig. 1A). The protein core formed by four dom ains is
slightly curved, giving rise t o a concave side harbouring
the N-terminal tail and a convex side with the putative
membrane-binding loops. The overall arrangement of the
individual helices shows some variation when compared
to AnxA5 and Anx24(Ca32), which is reflected by rather
large root mean square deviations (5.2–5.5 A
˚
for
alignment of C
a
atoms) of the structural superpositions.
In this overall structural fit, Anx(Gh1) differs significantly
even from another plant annexin, Anx24(Ca32). This
behaviour emphasizes the inherent flexibility of the
annexin fold, which nevertheless assembles both core
modules (domains I/IV and II/III) through the same

motifs seen in mammalian annexins (Table 2). The
intermodular salt bridge Glu113-Arg271 (IIB-IVB) is
conserved in both p lant annexins, as are the intramodular
salt bridges Asp93-Arg118 (IIB-IIC) and 276–280 (IVB-
IVC). Additionally, an interaction not seen in AnxA5 is
hydrogen bonding between CO117 and Arg276, thereby
tying together domains IIB, I VB and IVC.
The (artificially elongated) N-terminal tail consisting of
17 amino acids is visible in the current structure, apart from
the first three residues. The tail runs smoothly along the
concave surface of the protein and is anchored there by van
der W aals contacts (Leu9-Trp85) and by several hydrogen
bonds (CO10-His45, CO227-NH8, CO315-Thr8). In par-
ticular, the co ntact between CO10 and the iminium nitrogen
of His45, already identified in the structure of Anx24(Ca32),
seems to play an important role for the interaction between
core and N-terminal domain of plant annexins, since His45
is strictly conserved in t he plant subfamily.
As observed before with Anx24(Ca32), the globular
structure of Anx(Gh1), unlike that of mammalian annexins,
clearly shows separation of t he two modules ( I/IV and
II/III) leading to greater accessibility of the intermodular
space than in the case of AnxA5 and to formation of a
groove on the c onvex side (cf. Figure 1B). Located at the
entrance of the groove between domains III and IV is a
U-shaped, positively charged patch. The patch is formed
by five l ysine and three arginine residues ( Lys223, L ys226,
Arg238, Lys242, Lys249, Lys253, Arg256, and Arg291) and,
in the crystal structure, binds two sulfate ion s to compensate
for the excessive positive charge. The surface location in a

highly accessible area suggests that this U-shaped patch
might act as an electrostatic binding site for an interacting
protein that complements its geometry and charge. Addi-
tionally, the overall charge in this area might attract
negatively charged molecules and direct them into the
intermodular space where the putatively redox-active S
3
cluster is located (see below).
The IAB loop
In the IAB loop, Trp35 is strictly conserved and two
extreme conformations have been observed for this
residue in the crystal structure of Anx24(Ca32). In
the current structure, the conformation of the AB loop
in the fi rst domain d iffers from that of molecule 1 of
Anx24(Ca32) only around residues 33–37. Trp35 is found
in a surface exposed position and nestles into a rather
hydrophobic cleft presented by a symmetry-related mole-
cule oriented head-to-head. The exposed trypto phan side
chain is s andwiched between Arg261 and Tyr308, right
between the AB and DE loops of the domain IV of the
symmetry mate. Compared to Anx24(Ca32), the trypto-
phan residue in the present structure is s omehow halfway
between the loop-in and the loop-out position of the bell
pepper annexin.
Fig. 1. The three-dimensional structure of Anx(Gh1). (A) The fold of Anx(Gh1) as seen in a side view. Domain I is c oloured in dark blue, domain II
in light blue, domain III in aquamarine, a nd domain IV in green. Exposed surface residues on the convex side of t he molecule are explicitly drawn in
red. (B) Surface charge representation of the convex (left panel) and concave ( right panel) sides of the protein. Note the U -shaped , positively
charged patch between domains III and IV. This figure was prepared with
GRASP
[36].

Table 2. Conservation of salt bridges.
Anx(Gh1) Anx24(Ca32) AnxA5
IE-IIA Arg80-Glu99 – –
IIB-IVB Glu113-Arg271 Glu116-Arg272 Glu112-Arg271
IIB-IVB CO117-Arg276 CO120-Arg277 –
IIA-IIB Asp39-Arg118 Asp96-Arg121 Asp92-Arg117
IVB-IVC Arg276-Asp280 Arg277–281 Arg276-Asp280
2560 A. Hofmann et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Membrane binding loops
For reasons of homology, it is likely that the AB and DE
loops on the convex surface will serve as membrane binding
loops in the plant annexins, as was previously observed for
their mammalian relatives. In addition, the conservation of
aromatic and positively charged residues sticking out of the
convex surface (see Table 3) of plant annexins emphasizes
a possible functional role for membrane adsorption. Apart
from the loops IIIDE and the IVDE, all other membrane-
binding loops c arry conserved residues, which might either
interact with th e phospholipid headgroup or the glycerol
backbone region.
With respect to possible calcium binding in the membrane
loops, the recent crystal structure of Anx24(Ca32) raised the
question of how this might be accomplished by plant
annexins. As mentioned earlier, the endonexin sequence as a
constituent of canonical calcium binding sites in annexins is
conserved in domain I only and is present in a modified
form in the fourth domain. Despite extensive efforts, we
have not yet been successful in obtaining a calcium-bound
structure of Anx(Gh1) by soaking or cocrystallization
methods (data not shown). Analysis of possible molecular

mechanisms of calcium binding in plant annexins is
therefore restricted to homology modelling.
A comparison w ith AnxA5 as a template for canonical
calcium binding immediately shows t hat binding sites i n
domains II, III and IV are either distorted or the access of a
cation to the site is blocked by the p resence of a side chain
of a b asic residue. I n case of the IIAB s ite (Fig. 2), the
acidic re sidue a cting a s the bidentate ligand is substituted
by a histidine residue (His145 in the present structure),
which prevents a ccess to the binding site sterically and
electrostatically. It is noteworthy that this residue is strictly
conserved with plant annexins. Sites IIIAB and IVAB
show some distortion compared to the canonical confor-
mations and also contain positively charged residues with
repulsive effects a gainst cations. When looking at the s ites
in the first domain, however, it becomes clear that binding
of calcium is quite possible, in contrast to Anx24(Ca32).
Both IAB sites (Fig. 2) present a conformation ready
to accommodate a calcium ion, as does the low a ffinity
IDE site.
The carbonyls CO103, CO104 and the side chain of
Ser106 in the IIAB loop of Anx(Gh1) adopt a c onformation
that might be suited for coordination of a calcium ion,
although not in a canonical fashion. However, there is no
experimental proof for calcium binding in this location.
The S
3
cluster
Anx(Gh1) possesses four cysteine residues, two of which,
Cys116 and Cys243, belong to helices IIB and IIIE,

respectively. While positioned adjacent to each other, both
side chains exist in the reduced (thiol) form, although
formation of a disulfide bridge is sterically possible (Fig. 3).
This is even more remarkable since the protein was never
kept under reducing conditions. S imilar s ituations have
been observed i n other proteins, such as the fatty acid
binding protein [32] and cyclophilins [ 33]. The electron
density in this region c learly shows no additional p eaks,
which would indicate a dithioether linkage between both
side chains. The torsion angles N–C
a
–C
b
–S of the two
residues are )62° for Cys116 and )75° for Cys243, and the
sulfur atoms are separated by 5.5 A
˚
. As verified by
molecular modelling, a simple rotation a round the C
a
–C
b
cysteine side chain bonds would enable formation of a
dithioether linkage (N–C
a
–C
b
–S ¼ 65° for Cys116 and 102°
Table 3. Conservation of surface-exposed residues. Residue s in italic
indicate lack of conservation.

Location Anx(Gh1) Anx24(Ca32)
IAB Trp32 Trp35
IDE Lys72 Lys71
IIAB Arg103 Arg106
IIAB Trp104 Trp107
IIDE His144 Tyr147
IIDE His145 His148
IIIAB Lys187 Lys190
IIIAB Tyr189 Tyr192
IIIDE Lys230 Gly231
IVAB Arg261 Arg262
IVAB Arg262 Arg263
Fig. 2. The membrane binding loops. The IAB loops of Anx(Gh1) (A) and AnxA5 (B) are shown in the same view from the front. (C) and (D) show
the IIAB loops of Anx(Gh 1) and AnxA5, r espectively, from the top (membrane-binding) side. T he yellow ball indicates a calcium ion. Note tha t t he
IAB loop o f An x(Gh1) p rovides suitable environment for calcium binding. The IIAB loop, ho wever, featu res a histidine residue occluding acc ess to
the binding site. A bidentate ligand required for canonical calcium binding is also missing.
Ó FEBS 2003 X-ray structure of Anx(Gh1) (Eur. J. Biochem. 270) 2561
for Cys243, S–S distance: 2.2 A
˚
) with no other short
nonbonded interatomic contacts. Both cysteine residues are
conserved among several plant and mammalian annexin s,
among them annexin A2. In contrast to Anx(Gh1), the
structure of A nxA2 shows that both cysteine side chains
actually form a disulfide bridge [34].
Furthermore, the side chain of Met112 is positioned in
close vicinity and thereby enables formation of a triangular
sulfur cluster with distances between the sulfur atoms
ranging from 3.4 to 5.5 A
˚

. In their protonated forms, both
sulfhydryl groups interact with the methionine-S via
hydrogen bonding, establishing a 3S-2H topology with
almost tetragonal coordination on the methionine-S. This
S
3
cluster is located in the lower part of the annexin core in
module II/III and is acc essible only from the hydrophilic
cleft between modules I/IV and II/III, where Tyr250
provides shielding against direct interaction with solvent
molecules. Its plane is almost perpendicular to the S
3
plane
and the distance between the sulfur of Cys243 and the
tyrosine ring is 3.6 A
˚
.
While no experimentally proven chemical function of this
newly discovered cluster has been postulated so far, one can
easily imagine its involvement in the electron transfer
reactions. Oxidation of both cysteine residues to yield a
dithioether bond sets fr ee two electrons, w hich might be
donated to an oxidizing reagent, putatively a partly reduced
oxygen species. Hydrogen bonding of both sulfhydryl g roups
to the methionine certainly shifts the thiol-thiolate equili-
brium to the deprotonated side and therefore increases the
redox potential of the Cys2 system to more negative values.
Thus, Met112 acts as a factor to increase the red ox reactivity
of Cys116-Cys243. Tyr250 might be involved in these puta-
tive reactions by shuffling electrons from/to the S

3
cluster.
In this context, the finding of the unusual S
3
cluster in the
current structure presen ts a fascinating perspective for plant
annexin function, since it might well represent the structural
basis of the role of annexins in the oxidative stress response.
Oxy5, an annexin-like protein from Arabidopsis, was shown
to rescue E. coli DoxyR mutants and protect mammalian
cells from oxidative stress [8,9]. In particular, since the
constituting residues of the S
3
cluster are conserved in the
Arabidopsis protein (Fig. 4), it seems likely that this feature
forms the molecular basis of oxidative stress response by
these proteins. Similarly, an annexin from M. sativa was
reported to act as stress-response protein [7] and several
mammalian annexins are also known to be induced by a
variety of stress factors [5]. The U-shaped patch formed by
eight basic residues on t he entrance to the intermodular
groove on the convex side of the molecule might fu nction
to attract negatively charged partly reduced oxygen species
and direct them towards the redox active S
3
cluster, where
electrons fro m the cluster are used to reduce O()1) to O()2)
species.
Implications for cellulose synthase complex (rosette)
formation

Synthesis of b-1,4-glucan chains (cellulose) in plants requires
a chain elongation step during glucan polymerization,
which most likely is catalysed by cellulose synthase (CesA)
proteins. These proteins are components of plasma m em-
brane-bound CesA complexes w ith sixfold symmetry and
usually referred to as r osettes. Current models assume that
the active site of plant CesA proteins is formed on the
cytoplasmic face of t he plasma membrane by three Asp
Fig. 3. The sulfur cluster. Spatial arrangement of the S
3
cluster formed
by Met112, Cys116, and Cys243. The electron density shown was
calculated as omit map and is contoured at 1.5 r. Helices IIB and IIIE
are shown as Ca traces. Inset: T he distances between the individual
sulfur atoms are given in A
˚
.
Fig. 4. Amino acid sequence alignment. Aminoacidsequencesofdif-
ferent plant and mammalian annexins are aligned to show conserva-
tion of residues Met112, Cys116, Cys243, a nd Tyr250 of Anx(Gh1).
The sulfur-containing residues are marked red and the aromatic resi-
due (T yr or Phe) i s marked in cyan . All mammalian s equenc es shown
refer to the human proteins.
2562 A. Hofmann et al. (Eur. J. Biochem. 270) Ó FEBS 2003
residues together with a Q-X-X-R-W motif, both of which
are conserved. Eight transmembrane helices create a
channel through which the synthesized glucan chain is
secreted. The cytoplasmic N-terminal domain of CesA
proteins contains two zinc finger motifs, which recently have
been shown to bind zinc in a redo x-dependent manner (cf

[11] and references therein). W hile zinc binding occurs in the
reduced state of monomeric CesA protein, oxidation leads
to homo- or heterodimerization of CesA by formation of
intermolecular disulfide bonds (involving the Cys residues of
the zinc finger motif) and release of the metal ions. The
authors proposed a model [11] where the oxidized (dimer-
ized) state of CesA is required for rosette formation and
cellulose synthesis. The reduced (monomeric) state, how-
ever, is thought to be exposed to ubiquitin-moderated
degradation. As Anx(Gh1) has been colocalized with CesA
complexes [10], it is tempting to assume a role in the redox
mechanism of CesA, which presents t hree possibilities: (a)
Anx(Gh1) reduces (excessive) H
2
O
2
to H
2
O and acts as a
housekeeping protein; (b) Anx(Gh1) reduces intramolecular
disulfide bonds, which would rescue inactive CesA protein
for rosette formation; or (c) Anx(Gh1) reduces the inter-
molecular disulfide bonds of CesA, which leads to mono-
merization and thus inhibition of glucan synthesis. In an
earlier study [10], it was shown t hat Anx(Gh1) indeed
inhibits the activity of partially purified cotton fibre callose
synthase. In this context ( a) and (c) from above possible
models seem the most likely.
Conclusion
As reported i n the present s tudy, the three-dimensional

crystal structure of Anx(Gh1) from cotton emphasizes the
high conservation of the unique annexin fold even among
the members of the plant subfamily of annexin proteins. The
fold is comprised of the arrangement o f four a-helical
domains into two modules, which are held together by polar
interactions. Despite this overall conservation, the fold
allows for subtle differences, s uch as t he generation of a
groove on the convex side of the plant proteins, w hich is
not observed with non-plant an nexins s ince the modules
are packed much tighter.
A comparison of the current structure of Anx(Gh1) with
the structures o f Anx24(Ca32) and AnxA5 reveals that the
cotton annexin, in contrast to the bell pepper protein,
provides canonical calcium binding sites in the first domain.
The observed conformation of the other domains does not
allow binding of divalent c ations. The molecular mechanism
of calcium binding of plant annexins requires further studies
and work aimed at investigation of this matter is currently in
progress. The crystallization behaviour of Anx(Gh1) and
the results obtained in this study are certainly promising for
succeeding in determination of a calcium-bound structure of
a plant annexin.
A feature of particular interest in Anx(Gh1) is the
occurrence of two adjacent cysteine r esidues in helices IIB
and IIIE, which are observed in the present structure in
their reduced states, although formation of a dithioether
bond is possible by simple rotation a round the C
a
–C
b

bonds. Several mammalian annexins and even more plant
annexins show conservation o f these two cysteine residues
and some of them have been implicated in oxidative stress
response. Thus, it is very likely that this redox system forms
the basis of annexin response to oxidative stress in that it
reduces partly reduced oxygen species while being oxidized
to form a disulfide bridge. The presence of a nearby
methionine residue establishes an unusual sulfur cluster
with a 3S)2H topology. Hydrogen bonding is likely to
increase redo x r eactivity o f the Cys
2
system by increasing
the location probability of electrons at the thiolates, which,
in turn, will shift the redox potential of this system to less
negative values. A tyrosine residue in perpendicular con-
formation to the S
3
triangular plane is speculated to act as
electron carrier. This conclusion is supported by the fact
that the annexin-like protein Oxy5 from Arabidopsis shows
strict conservation in the constituting residues of the S
3
cluster, as well as the proximal tyrosine residue, and has
been proven experimentally to respond to oxidative stress.
Furthermore, the colocalization o f Anx(Gh1) with cotton
fibre cellulose synthase and its inhibiting effect on glucan
synthesis together with a recently discovered redox-depend-
ent dimerization of the chain elongation enzymes of
cellulose synthase strongly suggests a m odulatory role of
this annexin for cellulose synthase. Further studies to prove

this mechanism experimentally will be required.
Acknowledgements
We thank Zbigniew Dauter (NCI and NSLS, Brookhaven National
Laboratory) for help with data collection on beamline X9B and Robert
O. Gould and Malcolm Walkinshaw for helpful discussions.
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