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

This plant annexin consists of the typical annexin fold and is similar to the previously solved bell pepper annexin Anx24Ca32, but significant differences are seen when compared to the str

The crystal structure of annexin Gh1 from Gossypium hirsutum

Implications for cellulose synthase complex formation and oxidative stress response

Andreas Hofmann1, Deborah P Delmer2and Alexander Wlodawer3

1 Institute of Cell & Molecular Biology, The University of Edinburgh, Edinburgh, Scotland; 2 The Rockefeller Foundation,

New York, USA;3Macromolecular 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 to the final R-factor of 0.219 at

the resolution of 2.1 A˚ This plant annexin consists of the

typical
annexin fold 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 and II of Anx(Gh1) in their present conformation

All plant annexins possess a conserved 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 least five plant annexins and in several mammalian members of the annexin family of proteins, the methionine residue is conserved only

in three plant proteins Several of these annexins carrying the 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
2 ), hydroxyl radical (OHÆ), and hydrogen peroxide

(H2O2) These species 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 A6 [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 coliDoxyR mutants from H2O2stress Cotton 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 redox 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 distinct plant annexins occur most frequently and show 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 toA 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: Andreas.Hofmann@ed.ac.uk

(Received 7 February 2003, revised 20 March 2003,

accepted 8 April 2003)

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 molecule, 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 a priori

requirement that the side chains within the endonexin

sequence be conserved A different 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 adsorption, while the calcium harboured in

DE sites increases the binding affinity 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 in 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 is 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 system for these studies, we employed a different

plant protein, Anx(Gh1) from cotton, as a
prototype 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) and 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 in their reduced forms and a methionine residue The cluster is likely to be involved in redox reactions and might constitute the molecular basis

of oxidative stress response by annexins

Materials and methods Purification of recombinant protein Cloning and construction of an N-terminal His4-fusion protein has been described earlier [20] The 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 at 37C until the absorbance at

600 nm exceeded 1.0 Induction was carried out with 0.5 mM isopropyl thio-b-D-galactoside; at that time, 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 Ni2+-nitrilotriacetic acid column equili-brated with 100 mM NaCl, 20 mM Tris (pH 8.0) After extensive washing of the column, a stepwise elution protocol was performed with 20 mM, 50 mM, 100 mM, and 200 mM

imidazole in equilibration buffer The protein eluted at 50–100 mM 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 mMHepes (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 mM

Hepes (pH 8.0) Anx(Gh1) eluted at 230–350 mM 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 lL protein and 3 lL reservoir solution equilibrated against 300 lL reservoir solution at 285 K The crystals grew in about 8 weeks from 1.7M(NH4)2SO4, 0.1MHepes (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 mMCaCl2for between 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 mM in the presence and the absence of 1.4 mMphosphatidylcholine

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 k¼ 0.97950 A˚ Data were

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˚ Data processing was carried out with the

programsDENZOandSCALEPACK[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 a¼ b ¼ 61 A˚ and c ¼

215 A˚ Two-fold axes were detected parallel to [210] and

[120] in the self-rotation function calculated with GLRF

[22] This rendered P3112 and P3212 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 P3112 (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 coefficient [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 model of 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 withCNS 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 electron 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 throughout the procedure The structure was refined to the final R-factor of 0.219 (Rfree¼ 0.280) with reasonable overall geometry, as monitored with the programPROCHECK[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 oligomerization behaviour of plant annexins yielded calcium-independent monomer-trimer equilibria for annexins 23(Ca38), 24(Ca32), and Gh1, whereas Anx(Gh2) exists in a monomer-dimer equilibrium [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 collection

Number of independent reflections 27412

R merge 0.043 (0.433)

Refinement

No of reflections in working set/test set 24054 (3750)/

2656 (419)

Solvent statistics: number of water

molecules/sulfate ions

181/3

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

Structure of Anx(Gh1) in comparison with Anx24(Ca32)

and AnxA5

The three-dimensional crystal structure of annexin Gh1

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 domains is

slightly curved, giving rise to 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 Ca 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 plant 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, IVB 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 Waals contacts (Leu9-Trp85) and by several hydrogen

bonds (CO10-His45, CO227-NH8, CO315-Thr8) In

par-ticular, the contact 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 the plant subfamily

As observed before with Anx24(Ca32), the globular

structure of Anx(Gh1), unlike that of mammalian annexins,

clearly shows separation of the 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 convex 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 lysine and three arginine residues (Lys223, Lys226, Arg238, Lys242, Lys249, Lys253, Arg256, and Arg291) and,

in the crystal structure, binds two sulfate ions 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 S3 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 first domain differs 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 tryptophan side chain is sandwiched 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 somehow 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 coloured in dark blue, domain II

in light blue, domain III in aquamarine, and domain IV in green Exposed surface residues on the convex side of the 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 [36].

Table 2 Conservation of salt bridges.

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 carry conserved residues, which might either

interact with the 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 with AnxA5 as a template for canonical calcium binding immediately shows that binding sites in domains II, III and IV are either distorted or the access of a cation to the site is blocked by the presence of a side chain

of a basic residue In case of the IIAB site (Fig 2), the acidic residue acting as the bidentate ligand is substituted

by a histidine residue (His145 in the present structure), which prevents access 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 against cations When looking at the sites

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 affinity IDE site

The carbonyls CO103, CO104 and the side chain of Ser106 in the IIAB loop of Anx(Gh1) adopt a conformation 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 S3cluster

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 Similar situations have been observed in other proteins, such as the fatty acid binding protein [32] and cyclophilins [33] The electron density in this region clearly shows no additional peaks, which would indicate a dithioether linkage between both side chains The torsion angles N–Ca–Cb–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 around the Ca–Cb cysteine side chain bonds would enable formation of a dithioether linkage (N–C–C –S¼ 65 for Cys116 and 102

Table 3 Conservation of surface-exposed residues Residues in italic

indicate lack of conservation.

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(Gh1) and AnxA5, respectively, from the top (membrane-binding) side The yellow ball indicates a calcium ion Note that the IAB loop of Anx(Gh1) provides suitable environment for calcium binding The IIAB loop, however, features a histidine residue occluding access to the binding site A bidentate ligand required for canonical calcium binding is also missing.

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 annexins,

among them annexin A2 In contrast to Anx(Gh1), the

structure of AnxA2 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

S3cluster is located in the lower part of the annexin core in

module II/III and is accessible 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 S3plane

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 free two electrons, which might be

donated to an oxidizing reagent, putatively a partly reduced

oxygen species Hydrogen bonding of both sulfhydryl groups

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 redox reactivity

of Cys116-Cys243 Tyr250 might be involved in these

puta-tive reactions by shuffling electrons from/to the S cluster

In this context, the finding of the unusual S3cluster in the current structure presents 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 S3cluster are conserved in the Arabidopsisprotein (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 the entrance to the intermodular groove on the convex side of the molecule might function

to attract negatively charged partly reduced oxygen species and direct them towards the redox active S3cluster, where electrons from 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 mem-brane-bound CesA complexes with sixfold symmetry and usually referred to as rosettes Current models assume that the active site of plant CesA proteins is formed on the cytoplasmic face of the 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: The distances between the individual

sulfur atoms are given in A˚.

Fig 4 Amino acid sequence alignment Amino acid sequences of dif-ferent plant and mammalian annexins are aligned to show conserva-tion of residues Met112, Cys116, Cys243, and Tyr250 of Anx(Gh1) The sulfur-containing residues are marked red and the aromatic resi-due (Tyr or Phe) is marked in cyan All mammalian sequences shown refer to the human proteins.

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 redox-dependent manner (cf

[11] and references therein) While 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 three possibilities: (a)

Anx(Gh1) reduces (excessive) H2O2to H2O 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 that 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 in the present study, 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 of four a-helical

domains into two modules, which are held together by polar

interactions Despite this overall conservation, the fold

allows for subtle differences, such as the generation of a

groove on the convex side of the plant proteins, which is

not observed with non-plant annexins since the modules

are packed much tighter

A comparison of the current structure of Anx(Gh1) with

the structures of 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 cations 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 residues 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 around the Ca–Cb

bonds Several mammalian annexins and even more plant

annexins show conservation of 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 redox reactivity of the Cys2 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 S3triangular 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 S3 cluster, as well as the proximal tyrosine residue, and has been proven experimentally to respond to oxidative stress Furthermore, the colocalization of 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 modulatory 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. References

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