Crystal Structure of the Marasmius Oreades Mushroom Lectin in Complex with a Xenotransplantation Epitope

J. Mol. Biol. (2007) 369, 710–721

doi:10.1016/j.jmb.2007.03.016

Crystal Structure of the Marasmius Oreades Mushroom Lectin in Complex with a Xenotransplantation Epitope Elin Grahn 1 ⁎, Glareh Askarieh 1 , Åsa Holmner 1 , Hiroaki Tateno 2 Harry C. Winter 2 , Irwin J. Goldstein 2 and Ute Krengel 1 ⁎ 1

Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway 2

Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA

MOA, a lectin from the mushroom Marasmius oreades, is one of the few reagents that specifically agglutinate blood group B erythrocytes. Further, it is the only lectin known to have exclusive specificity for Galα(1,3)Galcontaining sugar epitopes, which are antigens that pose a severe barrier to animal-to-human organ transplantation. We describe here the structure of MOA at 2.4 Å resolution, in complex with the linear trisaccharide Galα(1,3) Galβ(1,4)GlcNAc. The structure is dimeric, with two distinct domains per protomer: the N-terminal lectin module adopts a ricinB/β-trefoil fold and contains three putative carbohydrate-binding sites, while the C-terminal domain serves as a dimerization interface. This latter domain, which has an unknown function, reveals a novel fold with intriguing conservation of an active site cleft. A number of indications suggest that MOA may have an enzymatic function in addition to the sugar-binding properties. © 2007 Elsevier Ltd. All rights reserved.

*Corresponding authors

Keywords: carbohydrate recognition; fungal lectin; mushroom agglutinin; protein–carbohydrate interactions; X-ray crystal structure

Introduction About 10% of the known species of mushrooms, or fruiting bodies of higher fungi, are regarded as edible, while a similar proportion is considered to be toxic to humans. Many species have been found to possess tonic and medical attributes. Numerous compounds with important pharmacological properties have been isolated from mushrooms and among these are several lectins. In recent years, mushroom lectins have become attractive tools owing to their distinct carbohydrate-binding speciPresent addresses: Å. Holmner, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden; H. Tateno, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-L71, La Jolla, CA 92037, USA. Abbreviations used: MOA, Marasmius oreades agglutinin; HUS, hemolytic-uremic syndrome; TTP, thrombotic thrombocytopenic purpura; r.m.s.d., root-mean-square difference/distance; PSL, Polyporus squamosus lectin; PDB, Protein Data Bank; GS I-B4, Griffonia simplicifolia isolectin I-B4; MalNEt, N-ethylmaleimide; PNGase, peptide:N-glycanase. E-mail addresses of the corresponding authors: [email protected]; [email protected]

ficities and their potential role in, e.g. blood group typing. Nevertheless, there is still very little known about the biological role of these proteins. Lectins are carbohydrate-binding proteins that are neither enzymes nor immunoglobulins. The synonym agglutinin originates from the ability of many lectins to agglutinate cells by presenting two or more binding sites on each molecule. Marasmius oreades agglutinin (MOA) is a lectin from the edible fairy ring mushroom. This protein has attracted scientific interest since the early 1950s, when it was first discovered to agglutinate human type B erythrocytes,1,2 which display the branched trisaccharide epitope Galα(1,3) [Fucα(1,2)]Galβ-epitopes.3 It has since been shown to have high affinity (in the micromolar range) for the so-called xenotransplantation antigens with terminal Galα(1,3)Galβ-epitopes. These epitopes are synthesized by a specific α1-3 galactosyltransferase that is expressed by all mammals except humans, apes and old-world monkeys.4 Consequently, the Galα(1,3)Galβ epitope is absent from human tissues and the presence of anti-Galα(1,3)Gal antibodies in human serum presents a major barrier to the use of porcine and other non-primate tissues and organs for xenotransplantation into humans. However, the reason behind xenograft failure and rejection has proven to be rather more complex than the mere incompatibility

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

X-ray Structure of MOA Lectin

of antigenic epitopes on xenotransplants. In cases where heart,5 and liver,6 from α1,3-galactosyltransferase knockout pigs were transplanted into baboons, graft survival was prolonged but inevitably led to necrosis. MOA is a suitable candidate for detection and isolation of blood group B structures in general, and xenotransplantation antigens in particular.7 The carbohydrate specificity of MOA resembles that of some bacterial toxins e.g. Escherichia coli verocytotoxin, which induces hemolytic-uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP), a severe human disease that can lead to thrombosis, kidney failure and cortical necrosis. Experiments have demonstrated that MOA can induce damage of the kidney glomerular epithelial cells in mice, which resembles the pathological patterns of HUS/TTP in humans,8 suggesting the application of MOA in a mouse model system for the human disease. To date, only eight X-ray crystal structures of mushroom lectins have been published. These proteins show great variation in their structures and carbohydrate specificities, indicating that there is no major conserved feature among the mushroom lectins. Here, we present the structure of MOA in complex with a xenotransplantation epitope. The Nterminal domain has a β-trefoil fold, as predicted from sequence comparisons. Two of the three potential binding sites (α and β) show clear electron density for the carbohydrate, and the presence of weak electron density in the third site indicates lessordered or weaker binding to the γ-site. The Cterminal domain, which is involved in dimerization, has an α/β-fold resembling the folds of some enzymes. A number of indications suggest that MOA may have enzymatic activity associated with its C-terminal domain.

Results The three-dimensional structure of MOA has been determined to 2.4 Å resolution in complex with the linear trisaccharide Galα(1,3)Galβ(1,4)GlcNAc. The electron density maps are of high quality (Figure 1(a)) and two trisaccharide ligands have been included in the model (Figure 1(b) and (c)). For the final model, Rfree is 21.1% and Rcryst is 19.0% (Table 1). The structure was determined in two different space groups, P21 and P3221. For the P3221 crystal form, which is the crystal form discussed in the following unless mentioned otherwise, each asymmetric unit contains one MOA protomer. Overall structure MOA adopts a dimeric structure, with the 2-fold axis coinciding with the crystallographic 2-fold axis (Figure 2(a)). Each protomer is composed of two distinct domains (Figure 2(b)). The N-terminal domain (residues 2–156) has a typical β-trefoil fold that is found in many lectins. This fold was first

711 observed in the ricin B-chain,9 and is therefore referred to as the ricin B-type lectin fold, or as the (QXW)3 fold because of the presence of three conserved Gln-X-Trp motifs. The N-terminal domain has a pseudo 3-fold repetitive symmetry, both in sequence and structure, with a basic motif of four β-strands repeated three times (β1-β4, β5-β8 and β9-β12) (Figure 2(b), (c) and (d)).10 Two βstrands from each motif are anchored in a sixstranded antiparallel β-barrel (β1, β4, β5, β8, β9 and β12), and the middle two strands (β2-β3, β6-β7 and β10-β11) extend from one side of the barrel in a hairpin-like fashion. The tryptophan side-chains of the three conserved QXW motifs meet at a pseudo 3fold axis in the center of this domain and contribute to the hydrophobic core. The C-terminal domain (residues 157–293) has an α/β-fold, which features a central six-stranded, mostly antiparallel, β-sheet (β14, β16–β20) flanked by three α-helices (α1–α3), and a short two-stranded β-sheet (β13, β15). The two domains are closely connected by a linker of approximately four residues in length. They further interact with each other via the loop connecting strands 3 and 4 of the N-terminal domain and via the loop following the third helix of the C-terminal domain. Dimerization MOA has been shown by size-exclusion chromatography to be a dimer.3 In both crystal forms described here, MOA adopts the same dimeric structure, which suggests that this is the biologically relevant molecule. The shape of the dimer is elongated with a maximum dimension of approximately 110 Å (Figure 2(a)). The dimer interface is mainly hydrophobic. It is formed by the third helix in the C-terminal domains of the two protomers. Upon dimer formation, a surface area of 1800 Å2 is buried on each subunit. This corresponds to 26% of the solvent-accessible surface area of the C-terminal domain, calculated using Areaimol.11 At both sides of the dimer interface, a tyrosine side-chain (Tyr180) is deeply immersed inside a pocket formed in the neighboring protomer, burying 96% of its accessible surface area of 195 Å2 (Figure 2(a)). Carbohydrate recognition MOA was co-crystallized with the trisaccharide Galα(1,3)Galβ(1,4)GlcNAc. MOA has three potential binding sites per protomer, referred to as α, β and γ, which are related by the pseudo 3-fold symmetry of the N-terminal β-trefoil domain. The three potential binding sites are formed by the three pairs of extended β-strands on one side of the protein, which assume the shape of shallow extended depressions on the protein surface. In two of the sites (α and β), well-ordered and clear density is present for each of the three individual sugar rings of the trisaccharide ligand, already in the original experimental electron density map (Figure 1(a)). In the third site (γ), however, only weak and discon-

712

X-ray Structure of MOA Lectin

Figure 1. A stereo representation of the MOA carbohydrate-binding site in complex with the trisaccharide Galα(1,3) Galβ(1,4)GlcNAc. (a) The sugar ligand (black) bound to the α-site and the residues important for sugar binding are shown as stick models. The experimental electron density map (Fobs) is contoured at 1.0 σ. (b) An overview of the protein–ligand interactions in the α-site. The trisaccharide (red) and the residues involved in direct and indirect hydrogen bonds (cutoff at 3.4 Å) are shown as stick models. Water molecules (W) are depicted as blue spheres. (c) A corresponding overview of the protein–ligand interactions in the β-site. This Figure as well as Figures 2(a), (b), (d), 3 and 4 were made using PyMoL [http://pymol.sourceforge.net/].

tinuous electron density was observed, and the ligand of the γ-site has, consequently, not been modeled. Yet, the presence of two patches of electron density, one between Trp138 and His148, and a weaker one extending towards Glu144, provide clear evidence that binding does occur, but rather weakly or with different specificity, compared to the α- and β-sites.

While binding of the saccharide ligand is clearly much stronger to the α and β-sites when compared to the γ-site, these two sites are not equivalent with respect to binding strength. The α-site shows the strongest electron density and is referred to here as the high-affinity binding site with occupancy 1.0, whereas the density in the β-site is somewhat weaker and the average occupancy of the ligand at

X-ray Structure of MOA Lectin

713

Table 1. Data collection and refinement statistics

A. Data collection Space group Cell parameters a (Å) b (Å) c (Å) α (deg.) β (deg.) γ (deg.) Wavelength (Å) Resolution (Å) Rmerge (%) I / σ(I) Completeness (%) Multiplicity B. Refinement Resolution (Å) No. reflections Rwork/Rfree (%) No. atoms Protein Ligand Water B-factors (Å2) Protein Ligand Water r.m.s deviations from ideal values Bond lengths (Å) Bond angles (deg.) Ramachandran plot Core region Outliers

Native

SeMet

P21

P3221

41.4 196.9 71.1 90 105.7 90

104.6 104.6 112.7 90 90 120 Remote 0.93952 70–2.4 (2.5–2.4) 8.9 (42.1) 21.3 (5.3) 100 (100) 10.6 (10.9)

1.0850 16–1.9 (2.0–1.9) 10.6 (35.4) 7.3 (1.7) 87.5 (84.1) 2.0 (1.9)

Peak 0.97905 90–2.4 (2.5–2.4) 9.3 (55.6) 19.1 (4.3) 100 (100) 10.8 (11.0)

19.8–2.4 26,661 19.0/21.1 2319 74 170 35.6 40.8 37.1 0.013 1.522 252 7

Values for the highest resolution shell are shown in parentheses. The Ramachandran plot analysis was performed for the nonglycine residues as described.40

this site has been refined to 0.8. The sugar bound to the β-site is also located slightly further away from the protein surface. When superimposing the two binding sites, the difference in distance between the two trisaccharides is 0.7–2.2 Å, with the Galαresidues being furthest apart (Figure 3(a)). There is, however, no difference in binding conformation of the ligands, which superimpose with a root-meansquare difference (r.m.s.d.) of 0.2 Å for all carbon atoms of the sugar-ring. Within each binding site, the electron density is stronger for the two galactose residues, compared to the N-acetyl glucosamine residue at the reducing end of the sugar. This observation is consistent with the fact that the majority of interactions are to the Galα(1,3)Galβ-epitope of the trisaccharide ligand (Figure 1(b) and (c); Table 2; and Supplementary Data Figure 1). In the α-site, the strongest hydrogen bonds are provided by Asp20, Asp23 and Thr38. Also Asn43 and His45 make significant contributions, by coordinating both the Galα 2OH and 3OH groups (Asn43) and the Galβ ring oxygen as well as the 4OH and 6OH groups (His45). Another residue of

central importance is Gln46, which is part of the conserved QXW motif, and indirectly anchors the sugar ligand at Galα 2OH and Galβ 4OH to several important structural elements in various parts of the protein sequence. The Galα 2OH and Galβ 4OH groups have been shown to be of critical importance for ligand recognition by MOA.12 Counterintuitively, the α-site contains the highest number of indirect water-mediated interactions. Important hydrophobic stacking interactions occur for the Galβ-residue only, involving mainly Trp35 and, to a lesser extent, His45. These residues pack against the Galβ-ring from opposite sides, but not in the typical parallel manner (Figure 1). It is interesting to note that the hydrophobic stacking interactions are absent for the terminal Galα residue, and that the 4OH group is coordinated only weakly by the lectin, both interactions that usually define galactose binding. Some of the interactions that are found in the αsite, such as those with Asp20, Asp23, Trp35 and His45, are conserved in the β-site, i.e. the interactions with Asp72, Asn75, Trp87 and His97. The interactions in the β-site are, however, slightly weaker, as estimated from the somewhat longer binding distances. The β-site, in addition, exhibits a number of significant differences compared to the αsite, despite a high level of sequence conservation (Figures 2(c) and 3(a)). Central to these differences is an interaction of Asp40 in the α-site with Lys265, a residue positioned in the C-terminal domain. This interaction does not have any counterpart in the β or γ-site. The two residues form a salt-bridge and, consequently, dislodge the whole loop region from residues 36 to 44 in the α-site (Figure 3(b)). Hence, at this position the α-site adopts a shape significantly different from that of the β and γ-sites (Figure 3(a); and Supplementary Data Figure 2). When superimposing the Cα atoms of the residues involved in formation of the three binding sites, i.e. residues 19– 47, 71–99 and 122–150 for the α, β, and γ-sites, respectively, the r.m.s.d. between the 29 superimposed Cα atoms is 1.3 Å when comparing the α and β-sites, 1.1 Å when comparing the α and γ-sites and 0.6 Å when comparing the β and γ-sites. In principle, the backbone atoms superimpose well for all three sites, with the exception of the loop formed by residues 36–44, 88–96 and 139–147 in the α, β and γ sites, respectively. In effect, even conserved residues like Thr38/90/141 are no longer equivalent (Figure 3(b)). For example, the position of the Thr38 side-chain in the α-site differs by 2.5 Å when compared to the corresponding side-chains of Thr90 and Thr141 in the β and γ-sites, respectively. The main consequences of the salt-bridge formed by Asn40 and Lys265 when comparing the sugar binding in the α- and β-sites are: (1) a much stronger coordination of the Galα 3OH group in the α-site compared to the β-site (where essentially no contact is found); and (2) a difference in coordination of the Galα(1,3)Galβ glycosidic bond oxygen atom, which interacts with Asn95 in the β-site, but is situated at a water-filled cavity in the α-site.

X-ray Structure of MOA Lectin

714 While the shapes and electrostatic potential of the β- and γ-sites are much more similar than those of the corresponding α-site (Supplementary Data Figure 2), there is one crucial exception: Glu144 of the

γ-site, which corresponds to Thr93 of the β-site and Thr41 in the α-site, points with its long and highly charged side-chain directly into the Galα recognition site, interfering with binding to the γ-site.

Figure 2 (legend on next page)

X-ray Structure of MOA Lectin

715

Figure 3. Comparison of the binding sites. (a) Superimposed backbone trace for the three binding sites α (blue), β (green) and γ (pink). The backbone trace is embedded in a surface representation of the α-site shown in light gray. For Asp40 and the residues exhibiting the largest differences in sugar binding, the side-chains are included in the picture. Likewise, the bound trisaccharides are shown as black (α-site) and grey (β-site) stick models. (b) A close-up view of the salt-bridge between Asp40 in the α-site loop and Lys265 from the C-terminal domain.

Crystal packing Due to the high solvent content of 77% (v/v) in crystals belonging to space group P3221, only a single contact site is found between MOA protomers caused by the crystal packing. One surface of this contact is formed mainly by the loops connecting β-strands 1 and 2, and β-strands 3 and 4 from the N-terminal domain together with αhelix 2 from the C-terminal domain. This surface interacts with the loops connecting β-strands 7 and 8, and β-strands 9 and 10 from the N-terminal domain of another MOA protomer. The GlcNAc residue of the trisaccharide bound to the β-site is also involved in this crystal packing interaction. The closest contact of this sugar residue to the neighboring molecule in the crystal is 3.0 Å (to Glu11). The α and γ-sites are not involved in any crystal packing interactions. The conformations of the three putative binding sites are equivalent in the two crystal forms, indicating that the crystal packing does not influence the sugar binding. Sequence and structure comparisons A search for homologous protein sequences gave only one hit for a close relative of MOA, a lectin from the mushroom Polyporus squamosus (PSL) which has 37% sequence identity with MOA.13 The carbohydrate specificity differs between the two proteins, with the PSL lectin being specific for sialylated glycoconjugates.14 No structure has been

published for PSL but, due to the high overall sequence similarity with MOA, we expect the two structures to be very similar. Only a few mushroom protein sequences are known, but it is likely that MOA/PSL-like proteins exist in other mushrooms as well. The final structural model of MOA was compared to other protein structures in the Protein Data Bank (PDB) using the DALI server.15 As expected, the DALI analysis indicated a high level of structural homology between the N-terminal domain of MOA and unrelated proteins containing β-trefoil domains e.g. hydrolases, cytokines, toxins and lectins. A DALI search with the MOA C-terminal domain gave hits with significantly lower Z-scores, indicating less similarity of this domain to other known structures in the PDB (Table 3). The C-terminal half of MOA adopts a fold that is often observed in catalytically active proteins such as N-glycanases, N-acetyltransferases, transglutaminases and cysteine proteases, which generally feature an active site cleft, in which the catalytic reaction takes place. When superimposing the top hit from the DALIsearch, a peptide:N-glycanase (PDB id 1X3W) with MOA, the proteins appeared to be quite different at first glance, but upon closer examination of the active site cleft, the similarity between the two proteins was striking (Figure 4(a) and (b)). In particular, the peptide:N-glycanase catalytic triad, Cys191, His218 and Asp235, superimposes very well with MOA residues Cys215, His257 and

Figure 2. Overall MOA structure. (a) The MOA dimer with the protomers colored in blue and green, respectively. The buried tyrosine side-chain of Tyr180 (blue/green) and the sugar ligands (black) are shown in stick representation. (b) A stereo image of one MOA protomer in schematic representation, showing the secondary structure elements colored from blue at the N-terminus to red at the C-terminus. The two modeled trisaccharide molecules are shown in black stick representation. α-Helices and β-strands are labeled. (c) The MOA sequence with secondary structure elements shown as arrows (red) for the β-strands and curls (black) for the α-helices. The carbohydrate-binding sites are marked by extended green bars and the conserved QXW motifs by blue boxes. The residues corresponding to amino acids of the putative catalytic triad are marked by arrowheads. This Figure was made using ESPript.42 (d) A view 90° rotated with respect to (b) around a horizontal axis, showing the three potential sugar-binding sites α, β and γ more clearly.

X-ray Structure of MOA Lectin

716 Table 2. Carbohydrate–protein interactions Ligand atom

Direct contact (Å)

GlcNAc C3 GlcNAc O3 GlcNAc C7 GlcNAc O7

3.8 / – 2.7 / 2.6 4.0 / – 3.4a / –

Interaction partner His45 Cε1 Asp23 Oδ1 Asp23 Cγ Asp23 Oδ2

Galβ C1 Galβ C2 Galβ O3

3.6 / – 3.9 / – – / 2.9 3.4 / –

His45 Cε1 / – His45 Cε1 / – – / Asn95 Nδ2 W1 / –

Galβ C4

3.8 / 3.6 3.8 / 3.7 – / 4.0 – / 4.0 2.7 / 2.7 – / 3.2 3.1 / 3.0 2.9 / – 3.7 / 3.6 3.8 / 3.7 2.9 / 3.4 3.7 / – 3.7 / 3.7 3.7 / 3.9 3.7 / 3.8 3.8 / 3.7 3.8 / 3.7 3.8 / 3.8 – / 4.0 2.8a / 3.1a – / 3.3a 2.5 / 3.1 3.2 / 3.1 3.6 / 3.5 2.8 / 3.0 2.7 / – – / 2.6

Trp35 Cε2 / Trp87 Cε2 Trp35 Cδ1 / Trp87 Cδ1 – / Trp87 Cδ2 – / Trp87 Cγ Asp20 Oδ2 / Asp72 Oδ2 – / Asn95 Nδ2 His45 Nδ1 / His97 Nδ1 W1 / – Trp35 Cε2 / Trp87 Cε2 Trp35 Cζ2 / Trp87 Cζ2 His45 Nδ1 / His97 Nδ1 Asp20 Cγ / – Trp35 Cε2 / Trp87 Cε2 Trp35 Cε3 / Trp87 Cε3 Trp35 Cδ2 / Trp87 Cδ2 Trp35 Cη2 / Trp87 Cη2 Trp35 Cζ2 / Trp87 Cζ2 Trp35 Cζ3 / Trp87 Cζ3 – / Tyr74 Cδ1 Asp20 Oδ1 / Asp72 Oδ1 – / Asp72 Oδ2 Asp23 N / Asn75 N His45 Nδ1 / His97 Nδ1 Trp35 Cδ1 / Trp87 Cδ1 Asn43 Nδ2 / Asn95 Nδ2 W1 / – – / W3

Galβ O4

Galβ C5 Galβ O5 Galβ C6

Galβ O6

Galα C1 Galα O2

Galα O3

Galα O4 Galα O5 Galα O6

2.7 3.2 3.1 3.1

/– /– /– /–

– / 3.1a 3.0 / 3.0 3.3 / 3.2

Indirect contact (Å)

Interaction partner

3.2 / – 2.9 / –

Trp44 N / – His45 N / –

– / 2.7 – / 2.6

– / Thr93 Oγ1 – / Gln98 Nε2

2.7 / – 2.7 / – 3.4 / –

Thr41 Oγ1 / – Ile42 O / – Asn43 Nδ2 / –

/– / Asn75 Nδ2 /– /–

Thr38 Oγ1 / – Thr38 N / – Asn43 Nδ2 / – W2 / – – / Gln88 Oε1 Trp35 Nε1 / Trp87 Nε1 Trp35 Nε1 / Trp87 Nε1

Hydrogen bonds involving oxygen or nitrogen atoms and hydrophobic carbon–carbon interactions with distances ≤3.4 Å and 4.0 Å, respectively, are listed. The bond lengths and interaction partners for the α-site are to the left and the corresponding information for the β-site is on the right side of the solidus (/). When a corresponding partner fulfilling the cut-off condition is missing, this is indicated by a dash (–). Water molecules involved in indirect carbohydrate–protein contacts are indicated by W. a Unfavorable angle for hydrogen bond.

Glu274. When comparing the peptide:N-glycanase catalytic triad with the corresponding residues in MOA, the r.m.s. difference for the three pairs of Cα atoms is 0.3 Å.16

Discussion The shortage of organs for transplantation into human patients provides the impetus for research into the possible use of animal organs for xenotransplantation, particularly from pigs. At present, however, this procedure is not practicable, due to the hyperacute immune response induced by the socalled xenotransplantation epitopes Galα(1,3)Galβ

and Galα(1,3)Galβ4(1,4)GlcNAc, ultimately leading to graft rejection.17 The most commonly used lectin specific for xenotransplantation epitopes is the B4 Griffonia simplicifolia isolectin (GS I-B4). This lectin specifically recognizes the terminal galactose residue of these epitopes and thus shares with many other lectins the property to recognize specific monosaccharides.18 MOA, however, has much lower affinity for monosaccharides and, instead, exhibits a binding preference for di- and trisaccharides. 3 The MOA structure provides an explanation for this binding property. In MOA, the binding site is not characterized by a deep pocket as present in the GS I-B4 structure,18 but instead forms an extended shallow

X-ray Structure of MOA Lectin

717

Table 3. Top-five hits from DALI search, done with the C-terminal domain of MOA PDB id

Z

1X3W

5.3

3.4

92

317

10

1FL3

4.3

3.0

92

721

10

2F4O

4.1

3.5

93

290

8

1CV8

4.1

2.9

81

173

15

1XKG

4.0

3.1

93

298

8

RMSD LALI LSEQ %IDE

Protein Peptide N-glycanase (hydrolase) Plasma factor (transglutaminase) Peptide N-glycanase (hydrolase) Staphopain (cysteine proteinase) Allergen Der p 1 (hydrolase)

The following notation is used for the data in the Table: PDB id, PDB accession code of the search and aligned structure; Z score, a measure of the strength of structural similarity in units of standard deviation; RMSD, positional root-mean-square deviation of the superimposed Cα atoms; LALI, total number of equivalenced amino acid residues; LSEQ, length of the entire chain of the equivalenced structure; %IDE, the percentage of sequence identity over equivalenced positions.

groove on the protein surface. All the typical interactions characteristic for galactose recognition, such as hydrophobic stacking interactions with aromatic protein side-chains and a strong coordination of the 4OH group are missing for the terminal galactose residue in the MOA structure. Instead, the protein exhibits similar interactions to the adjacent galactose residue, Galβ. Accordingly, MOA has higher specificity for terminal Galα(1,3) Galβ epitopes, while GS I-B4 is less specific and recognizes a broader spectrum of terminal Galα epitopes. Recent studies have shown MOA to be superior to GS I-B4 lectin in recognizing the porcine xenotransplantation antigen, since it shows significantly higher selectivity for the Galα(1,3)Galβ, epitope.19 The binding affinity is increased further by the addition of an α-2-linked L-fucose molecule to the Galβ residue, as in the branched blood group B trisaccharide Galα(1,3)[Fucα(1,2)]Galβ. 3,12,20 Manual docking of the blood group B trisaccharide into the binding sites (not shown) is easily achieved, as both the α and β-sites are solventexposed. Binding is expected to be particularly strong in the α-site, with Trp44 serving as potential stacking partner, whereas the corresponding residue in the β-site is Pro96. When comparing the MOA structure with the only other available mushroom lectin structure with a β-trefoil fold, the Laetiporus sulphureus lectin (PDB id 1W3F)21 the β-trefoil domains of the structures were found to superimpose well with an r.m.s.d. of 1.5 Å, even though there is no similarity in sequence. This feature of having a high level of structural similarity despite a low level of sequence conservation is a well-known property of β-trefoil proteins (Figure 4(c)).10 A comparison with the other seven mushroom lectin structures known to date reveals even lower levels of similarity both with respect to sequence conservation and to the overall threedimensional fold.

The MOA structure has three distinct binding sites, which are related by 3-fold pseudo symmetry. These are characterized by multiple interactions, and the majority of the residues critical for binding are conserved in all of them. All sites appear to bind the Galα(1,3)Galβ4(1,4)GlcNAc ligand, but with different degrees of affinity. The α and β-sites display tighter binding than the γ-site, into which the ligand was chosen not to be modeled. These observations were unexpected in light of an earlier mutagenesis study on MOA, in which 20 mutations positioned in the binding site-regions were probed for binding. Only substitutions at Gln46 (α-site) and Trp138 (γ-site) and a C-terminal truncation were reported to have a significantly negative effect on binding avidity,22 which led to the conclusion that only the α and γ-sites were binding-active. On the basis of our structural observations, we believe that the single mutations introduced at the β-site had little effect on binding avidity, due to the compensatory effects of multiple interactions provided by other essential residues at this site. On the other hand, in the γ-site the effect of the single substitution at position 138 (W138A) had great impact on binding because of the loss of the essential stacking interaction. Explaining the dramatic effect of the Q46A mutation22 is somewhat more difficult on the basis of structural data alone. The reduced binding activity caused by this substitution could have resulted from the loss of the important watermediated interactions to the Galα 2OH and Galβ 4OH groups (Figure 1(b); and Supplementary Data Figure 1(a)). However, Gln46 is involved also in several intramolecular interactions, which contribute to the stability of the binding site and are not observed in the β-site. Furthermore, the critical interactions with the hydroxyl groups 2OH and 4OH in the β-site are provided by Asn95 (Figure 1(c); and Supplementary Data Figure 1(b)), a residue that was not tested in the mutagenesis study. The marked reduction in binding avidity of the truncated lectin may be caused simply by the loss of the C-terminal domain, which acts as a dimerization platform in the protein. One protomer will have only three available binding sites, i.e. half as many as in the dimer, where all of the six sites are positioned on the same side of the protein. In addition, the salt-bridge between Lys265 and Asp40 positioned in the α-site binding loop will be lost, an interaction that most likely contributes to the integrity of the high-affinity binding site. In summary, when the overall binding avidity is measured, it is difficult to estimate the individual affinity contribution of each binding site, and the strength and specificity of the carbohydrate–protein interactions are even harder to trace back to single-point mutations. While the carbohydrate-binding specificity of MOA is well characterized,3,12,20,22 little is known about the biological function of MOA in the mushroom, other than that it appears to be a cytosolic protein, since it lacks a signal sequence. The only published report discussing the function

718

X-ray Structure of MOA Lectin

Figure 4. Structural analysis of MOA. (a) Superimposition of MOA in rainbow colors and the top hit from the DALI search, a peptide:N-glycanase, PNGase (PDB id 1X3W) shown in grey. The grey box marks the section enlarged in (b). (b) A close-up view of the superimposition at the PNGase active site cleft, showing the side-chains of the PNGase catalytic triad, Cys191, His218 and Asp235, and the corresponding MOA residues, i.e. Cys215, His257 and Glu274 for the P21 crystal form. (c) The superimposition of the two mushroom lectins with β-trefoil folds, MOA and Laetiporus sulphureus lectin (PDB id 1W3A), in green and black, respectively. The ligands modeled into the α and β-sites of MOA are shown as green stick models. Despite the apparent lack of sequence homology, the overall folding topology of the ricin-like lectin domains is highly conserved. (d) A potential active site cleft of MOA. Superimposition of the trigonal (orange-yellow) and monoclinic (green) crystal structures of MOA. The electron density is shown for the three residues Cys215, His257 and Glu274 of the P3221 crystal form. The corresponding side-chains are shown as thin green sticks for the P21 crystal form. The extra density observed next to Cys215 (encased by a grey box) is likely caused by a bound MalNEt molecule, which was used in the crystallization setup from which the P3221 crystal was obtained. Note that the side-chain of His257 in the P21 structure, which was crystallized in the absence of MalNEt, points right into the putative MalNEt density.

of this lectin shows that MOA induces renal thrombotic symptoms, both in vivo if injected into mice intravenously and in vitro, with pathological patterns of thrombotic microangiopathic lesions that are similar to those induced by bacterial

toxins.8 The bacterial toxins that induce HUS/ TTP, e.g. E. coli verocytotoxin (AB5 toxin) and toxin A from Clostridium difficile, share the binding specificity with MOA, but differ greatly in structure and function. There are, however, several other

X-ray Structure of MOA Lectin

lectins with cytotoxic properties, e.g. ricin,9 the mistletoe lectins,23,24 the abrin lectin,25 the sea cucumber lectin,26 and the L. sulphureus mushroom lectin,21 which share the β-trefoil fold and show close structural similarities with the MOA Nterminal domain. Some of these proteins exhibit a hemolytic pore-forming module, 21,26 while others are coupled to enzymatic activities, e.g. related to ribosome inactivation.9,23,24,25 Currently, there is no evidence that the natural biological function of MOA is coupled to toxic activity; what is intriguing, however, is the apparent resemblance between the C-terminal domain of MOA and various catalytically active proteins, such as certain glycanases and proteases. This structural similarity is especially apparent in the active site cleft, and extends to the great similarity of the catalytic triad as well as to several additional important residues in the cleft of these enzymes. Furthermore, in the P3221 crystal structure reported here, there is an additional patch of electron density attached to the side-chain of Cys215 that could be part of the putative catalytic triad (Figure 4(d)). Since Nethylmaleimide (MalNEt) was present as an additive in the crystallization solution, and MalNEt is known to be able to covalently bind free cysteine, we assume that this extra density originates from a MalNEt molecule bound to Cys215. This finding suggests that the Cys215 side-chain is highly accessible, and that its thiol group is reactive, which makes Cys215 a candidate to participate in a catalytic reaction. Experiments are in progress to discover if MOA indeed has enzymatic activity as suspected and, if so, which reaction it catalyzes. In conclusion, the structure of MOA in complex with the xenotransplantation epitope Galα(1,3) Galβ(1,4)GlcNAc reveals detailed characteristics of its noteworthy sugar-binding properties and provides new insights into the function of this protein. We find convincing evidence that MOA may have enzymatic properties associated with its C-terminal domain, a property that requires further study. This structural investigation provides a solid base for future research, concerning important potential applications of the MOA protein as an immunodiagnostic reagent and in a mouse model system for the human HUS/TTP syndrome, and sheds new light on the biological role of a mushroom lectin, a field that has so far been very little studied.

Materials and Methods Expression and purification Native and SeMet-labeled MOA were expressed recombinantly in E. coli and purified as described.3,27 SeMetlabeled protein was obtained by expression under suppression of the methionine pathway.28,29 The purified protein was dialyzed against water and lyophilized. For crystallization, the MOA protein was dissolved in 20 mM Tris–HCl (pH 7.5) to a protein concentration of 10 mg/ml

719 and mixed with the trisaccharide Galα(1,3)Galβ(1,4) GlcNAc (Dextra Laboratories Ltd., Readings, UK) at a molar ratio of 5:1, before setup. Crystallization The trigonal MOA crystals were obtained as described.30 The crystal used for SeMet- multiple anomalous dispersion (MAD) data collection was grown from the following conditions: 0.1 M Hepes (pH 6.5), 2.4 M ammonium formate and 0.6 mg/ml MalNEt (Sigma Aldrich) at 293 K. The crystal had dimensions of 0.2 mm × 0.2 mm × 0.1 mm. Due to earlier problems with poor Se-incorporation (indicated by mass spectrometry and intensity variation of the Se signals in EXAFS selenium scans between crystals), the crystal was also soaked with heavy atoms, applying the following procedure: The crystal was first transferred to a 10 μl drop of mother liquor. Subsequently, a few grains of sodium tungstate dihydrate (Hampton Research, USA) were added to the opposite side of the drop, compared to the location of the crystal. The crystal was observed under the microscope while the tungstate salt dissolved, after 20 min, the crystal was transferred to a 5 μl drop containing mother liquor plus 30% (v/v) glycerol for back-soaking and cryo-protection. After 1 min, the crystal was plunged into liquid nitrogen and stored at 100 K. Analysis of the collected diffraction data (and subsequent phasing) identified the space group to be P3221, with one protomer per asymmetric unit and cell parameters of a = b = 104.6 Å, c = 112.7 Å, α = β = 90° and γ = 120°. Using the hanging-drop vapor-diffusion technique, we have also obtained crystals in an alternative crystal form not previously published. Thin, rod-shaped crystals grew from the following conditions: 0.1 M Tris–HCl (pH 8.5), 25–30% (w/v) PEG (1000, 2000 or 4000 Da) and 2% (v/v) 2-propanol. The crystals appeared after one week compared to the three months required for the P3221 crystal form. A native dataset was collected for a crystal grown from 0.1 M Tris–HCl (pH 8.5), 30% PEG 1000, 2% 2-propanol and 6% dimethyl sulfoxide. The dimensions of the crystal were approximately 0.1 mm × 0.03 mm × 0.03 mm. Glycerol was added on top of the crystallization drop before fishing the crystal and flash-freezing it in liquid nitrogen. A dataset was collected at beamline I711, Max-labII, Lund, Sweden, using a Marresearch 165 mm CCD detector. A total of 250 images were collected with an oscillation angle of 0.5°. Analysis of the diffraction data were consistent with space group P21 with four protomers per asymmetric unit and cell parameters of a = 41.4 Å, b = 196.9 Å, c = 71.1 Å, α = γ = 90° and β = 105.7°. The Matthews coefficient is 2.1 Å3/Da, which corresponds to a solvent content of 42% (v/v). Data collection, processing, scaling and structure determination For the SeMet labeled P3221 crystal, one dataset was collected at the Se-peak (12.6638 keV, corresponding to a wavelength of 0.97905 Å) and one at a remote wavelength (13.2 keV, 0.93952 Å), at beamline ID14-4, ESRF, Grenoble, France. An additional dataset was collected at the tungsten peak (10.212 keV, 1.21414 Å); however, these data were not used for further analysis, since the structure could be solved using the Se-data alone. A total of 180 images were collected for each dataset, using an oscillation angle of 1° at a temperature of 100 K on a Q315r ADSC

X-ray Structure of MOA Lectin

720 CCD detector. The data were less anisotropic compared to those obtained previously.30 After processing with XDS,31–33 and MOSFLM,34 the data were scaled, using SCALA.35,36 The structure was solved with the multiple anomalous dispersion phasing method, using SHELX.37 Five Se-positions were identified, corresponding to five methionine residues out of 292 residues in each protomer. The Matthews coefficient is 5.4 Å3/Da, which corresponds to a solvent content of 77% (v/v). The scaling statistics are presented in Table 1.

beamline, ESRF, Grenoble, France and the staff at MAX-lab in Lund for assistance during data collection of the second MOA data set. This work has been supported by grants from EMBIO (postdoctoral research position of E.G.), the Norwegian Research Council (grant 171631/V40; to U.K.) and the NIH (grant GM-29470; to I.J.G.).

Supplementary Data Model building and refinement The initial electron density map was of excellent quality and could be readily interpreted. All amino acid residues were built into the electron density in a single step, using Coot,38 followed by refinement of the structure using REFMAC5.39 Subsequently, the carbohydrate ligands were introduced into the model in two of the three binding sites. The occupancies of the two trisaccharide ligands were refined to match the average B-factors of the coordinating protein residues. Final occupancies are 1.0 for the α-site and 0.8 for the β-site ligand, consistent with the weaker electron density at the latter site. Water molecules were added using REFMAC5,39 and were visually inspected using Coot before acceptance. 38 Methionine residues were not replaced by selenomethionine residues, since SeMet incorporation was incomplete, with occupancies refined to values significantly below 1.0. The final model contains residues 2–293, two trisaccharide molecules and 170 water molecules. The average real space correlation coefficient is 93% for the experimental map. There was no electron density at all for the first methionine residue, indicating that it might be cleaved off during protein synthesis. The Ramachandran plot, obtained using MOLEMAN2,40 shows 2.7% outliers, but most of these eight residues lie close to allowed areas, with the exception of residues 106, 211 and 286. These are found in loop-regions with main-chain real space correlation coefficient values of 86%, 93% and 81%, respectively. The structure of the P21 crystal form was solved by molecular replacement using the program MOLREP,41 with two MOA-dimers as a search model. A complete model refinement was carried out, however, only for the P3221 crystal form, since the electron density maps were of higher quality, despite the lower resolution. The P21 crystal structure was used mainly to confirm that the dimer formation was not a crystal artefact and to compare the structural details at the potential active site with and without MalNEt. Refinement statistics are summarized in Table 1. Protein Data Bank accession code Coordinates and structure factors for the refined MOA model in space group P3221 have been deposited at the Protein Data Bank with the accession code 2IHO.

Acknowledgements We thank Edward Mitchell and Andrew McCarthy for excellent technical assistance at the ID14-4

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2007.03.016

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Edited by I. Wilson (Received 15 November 2006; received in revised form 2 March 2007; accepted 3 March 2007) Available online 15 March 2007