The crystal structure of annexin Gh1 from Gossypium hirsutum reveals an unusual S3 cluster

Implications for cellulose synthase complex formation and oxidative stress response

Authors


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: Andreas.Hofmann@ed.ac.uk

Abstract

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 Å. 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 mammalian annexin AnxA5 indicates that canonical calcium binding 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 Å, 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.

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 (inline image), hydroxyl radical (OH·), 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 Arabidopsis 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 coliΔoxyR mutants from H2O2 stress. 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 characterized, 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 (mallow), 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 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 carbonyl 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 membrane-binding side. When comparing the membrane-binding 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-dimensional 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 mg·L−1 ampicillin) were inoculated with an overnight culture of 1 L. The cells were grown at 37 °C until the absorbance at 600 nm exceeded 1.0. Induction was carried out with 0.5 mm isopropyl thio-β-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 equilibrated 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 purified by anion exchange chromatography with Q-sepharose. Pooled fractions obtained by affinity chromatography were diluted threefold with 20 mm 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 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 µL protein and 3 µL reservoir solution equilibrated against 300 µL reservoir solution at 285 K. The crystals grew in about 8 weeks from 1.7 m (NH4)2SO4, 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 mm CaCl2 for 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 concentrations of 2–15 mm in the presence and the absence of 1.4 mm phosphatidylcholine.

X-Ray data collection and structure solution

While diffraction obtained from crystals with the in-house equipment was limited to 3.2 Å, the maximal resolution achieved at the synchrotron beamline X9B (NSLS, Brookhaven National Laboratory) was 2.1 Å. The structure was solved using the synchrotron data set GH1–3A4, which was collected at a wavelength λ = 0.97950 Å. 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 Å were recorded, while in the second run the detector was moved closer to the crystal to record reflections between 8 and 2.1 Å. Data processing was carried out with the programs denzo and scalepack[21] and the data collection statistics are summarized in Table 1.

Table 1. Data collection and refinement statistics. Values for the last resolution shell (2.23–2.10 Å) are given in parentheses.
Data setGH1–3A4
  1. a Estimated from scalepack output; b Rfree defined in [35].

Data collection
 Space groupP3112
 Cell dimensions (Å)61.05, 61.05, 215.36
 Resolution (Å)40–2.1
 Number of measurements808320
 Number of independent reflections27412
 Completeness100% (100%)
 Multiplicitya7
 Rmerge0.043 (0.433)
Refinement
 No of reflections in working set/test set24054 (3750)/ 2656 (419)
 Visible residues4–321
 Number of non-H atoms2549
 Solvent statistics: number of water molecules/sulfate ions181/3
 R/Rfreeb0.219 (0.300)/ 0.280 (0.377)
 Average B-factor for all atoms (Å2)51.9
 Ramachandran plot: Residues in most favoured/additionally allowed/generously allowed region (%)87.5/11.5/1.0

The diffraction pattern indicated a trigonal space group with approximate cell dimensions of a = b = 61 Å and c = 215 Å. 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 σ, 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 with cns v. 1.0 [26] employing 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 (Rfree = 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 generating 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.

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 Å for alignment of Cα 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.

Figure 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 grasp[36].

Table 2. Conservation of salt bridges.
 Anx(Gh1)Anx24(Ca32)AnxA5
IE-IIAArg80-Glu99
IIB-IVBGlu113-Arg271Glu116-Arg272Glu112-Arg271
IIB-IVBCO117-Arg276CO120-Arg277
IIA-IIBAsp39-Arg118Asp96-Arg121Asp92-Arg117
IVB-IVCArg276-Asp280Arg277–281Arg276-Asp280

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 particular, 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. Additionally, 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 molecule 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 tryptophan residue in the present structure is somehow halfway between the loop-in and the loop-out position of the bell pepper annexin.

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.

Table 3. Conservation of surface-exposed residues. Residues in italic indicate lack of conservation.
LocationAnx(Gh1)Anx24(Ca32)
IABTrp32Trp35
IDELys72Lys71
IIABArg103Arg106
IIABTrp104Trp107
IIDEHis144Tyr147
IIDEHis145His148
IIIABLys187Lys190
IIIABTyr189Tyr192
IIIDELys230Gly231
IVABArg261Arg262
IVABArg262Arg263

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 conformations 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.

Figure 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.

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 S3 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. 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–Cα–Cβ–S of the two residues are −62° for Cys116 and −75° for Cys243, and the sulfur atoms are separated by 5.5 Å. As verified by molecular modelling, a simple rotation around the Cα–Cβ cysteine side chain bonds would enable formation of a dithioether linkage (N–Cα–Cβ–S = 65° for Cys116 and 102° for Cys243, S–S distance: 2.2 Å) 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].

Figure 3.

The sulfur cluster. Spatial arrangement of the S3 cluster formed by Met112, Cys116, and Cys243. The electron density shown was calculated as omit map and is contoured at 1.5 σ. Helices IIB and IIIE are shown as Ca traces. Inset: The distances between the individual sulfur atoms are given in Å.

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 Å. 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 S3 cluster 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 S3 plane and the distance between the sulfur of Cys243 and the tyrosine ring is 3.6 Å.

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 equilibrium 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 putative reactions by shuffling electrons from/to the S3 cluster.

In this context, the finding of the unusual S3 cluster 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. coliΔoxyR mutants and protect mammalian cells from oxidative stress [8,9]. In particular, since the constituting residues of the S3 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 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 S3 cluster, where electrons from the cluster are used to reduce O(−1) to O(−2) species.

Figure 4.

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

Implications for cellulose synthase complex (rosette) formation

Synthesis of β-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 membrane-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 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 (dimerized) state of CesA is required for rosette formation and cellulose synthesis. The reduced (monomeric) state, however, 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) H2O2 to 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 intermolecular disulfide bonds of CesA, which leads to monomerization 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 α-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 Cα–Cβ 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 conformation to the S3 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 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-dependent 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.

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