Gliotoxin Biosynthesis: Structure, Mechanism, and Metal Promiscuity

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Gliotoxin biosynthesis: Structure, mechanism and metal promiscuity of carboxypeptidase GliJ Antoine Marion, Michael Groll, Daniel H. Scharf, Kirstin Scherlach, Manuel Glaser, Holger Sievers, Michael Schuster, Christian Hertweck, Axel A. Brakhage, Iris Antes, and Eva M. Huber ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

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ACS Chemical Biology

Gliotoxin biosynthesis: Structure, mechanism and metal promiscuity of carboxypeptidase GliJ Antoine Marion‡, Michael Groll§, Daniel H. Scharf||,†, Kirstin Scherlach#, Manuel Glaser‡, Holger Sievers⊥, Michael Schuster⊥, Christian Hertweck#,¶, Axel A. Brakhage||,^, Iris Antes‡,* and Eva M. Huber§,* ‡

Center for Integrated Protein Science Munich at the Department of Biosciences, Technische Universität München, Emil-Erlenmeyer-Forum 8, D-85354 Freising, Germany

§

Center for Integrated Protein Science Munich at the Department Chemistry, Technische Universität München, Lichtenbergstr. 4, D-85748 Garching, Germany

||

Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), D-07745 Jena, Germany ⊥Fachgruppe

Analytische Chemie, Technische Universität München, D-85748 Garching, Germany

#

Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), D-07745 Jena, Germany ¶ ^

Chair of Natural Product Chemistry, Friedrich Schiller University (FSU), D-07743 Jena, Germany

Department of Microbiology and Molecular Biology, Friedrich Schiller University (FSU), D-07743 Jena, Germany

ABSTRACT: The formation of glutathione (GSH) conjugates, best known from the detoxification of xenobiotics, is a widespread strategy to incorporate sulfur into biomolecules. The biosynthesis of gliotoxin, a virulence factor of the human pathogenic fungus Aspergillus fumigatus, involves attachment of two GSH molecules and their sequential decomposition to yield two reactive thiol groups. The degradation of the GSH moieties requires the activity of the Cys-Gly carboxypeptidase GliJ, for which we describe the X-ray structure here. The enzyme forms a homodimer with each monomer comprising one active site. Two metal ions are present per proteolytic center, thus assigning GliJ to the diverse family of dinuclear metallohydrolases. Depending on availability, Zn2+, Fe2+, Fe3+, Mn2+, Cu2+, Co2+ or Ni2+ ions are accepted as cofactors. Despite this high metal promiscuity, a preference for zinc versus iron and manganese was noted. Mutagenesis experiments revealed details of metal coordination and molecular modeling delivered insights into substrate recognition and processing by GliJ. The latter results suggest a reaction mechanism in which the two scissile peptide bonds of one gliotoxin precursor molecule are hydrolyzed sequentially and in a given order.

INTRODUCTION Aspergillus fumigatus is the main causative organism of invasive aspergillosis, a life-threatening disease for immunocompromised patients.1 One of its virulence factors is the secondary metabolite gliotoxin, the prototype of epipolythiodioxopiperazine (ETP) compounds.2-4 Apart from its diketopiperazine scaffold, which is nonribosomally synthesized from the amino acids L-Phe and 5-7 L-Ser, gliotoxin features a reactive disulfide bond that is vital for its toxicity.8 The epidithiobridge is enzymatically installed by oxidation of the free dithiol precursor.9-11 The sulfur atoms originate from two glutathione (GSH) molecules12 that are sequentially degraded to the dithiol intermediate upon addition to the diketopiperazine moiety (Figure 1).13, 14 Attachment of GSH and its subsequent stepwise dissection is a commonly employed detoxifica-

tion strategy for xenobiotics in plants and animals. In addition, GSH conjugation and decomposition is known from the biosynthesis of plant defense metabolites such as glucosinolates and the indole alkaloid camalexin15-18. The degradation of the bis-glutathionediketopiperazine adduct formed during gliotoxin production in A. fumigatus requires three enzymes, one of which, GliJ, cleaves the Cys-Gly peptide bonds once the γ-glutamyl residues have been removed by GliK (Figure 1). GliJ displays about 40% sequence identity to human renal dipeptidase (HRD), a glycoprotein responsible for glutathione metabolism, the conversion of leukotrienes and the hydrolysis of β-lactam antibiotics (Supplementary Figure 1).19 HRD is described as a metalloprotease featuring a binuclear Zn2+ cluster in its active site albeit lacking sequence motifs typical of zinc proteases.20, 21 The primary

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Figure 1. The biosynthesis of gliotoxin. Gliotoxin is synthesized from the proteinogenic amino acids L-Phe and L-Ser and two molecules of glutathione (GSH; upper panel). Condensation of L-Phe and L-Ser followed by attachment of two GSH molecules yields a bis-glutathione conjugate. This intermediate undergoes sequential enzymatic degradation (lower panel). GliK removes the γ-glutamyl moieties, the carboxypeptidase GliJ, the subject of this study, cleaves the Cys-Gly dipeptide bonds and the pyridoxal 5’-phosphate dependent β-lyase GliI creates a dithiol intermediate. Further conversions finally yield the biologically relevant disulfide-bridged version of gliotoxin. differential scanning fluorimetry assays prove that the Cterminal tail of GliJ significantly contributes to the stability of the protein (Supplementary Figure 4B) and molecular modeling data predict a crucial role of the C-terminal residues in substrate recognition (see below and Figure 3).

sequences of GliJ and HRD are not related to other metallo19 enzymes but are similar to a cluster of ~400 microbial dipeptidases, for which Sco3058 from Streptomyces coelicolor is 22 the best characterized representative. In comparison to HRD and Sco3058, we here investigated A. fumigatus GliJ from a structural and mechanistic point of view using X-ray crystallography, mutagenesis, molecular modeling as well as analytical and biochemical methods.

The active site. GliJ features two solvent-accessible active sites, one per monomer, separated by ~40 Å. Each proteolytic center has two metal ions bound. TXRF (total reflection Xray fluorescence) measurements detected high concentrations of iron in purified GliJ samples compared to buffer controls (Supplementary Figure 5 and Supplementary Table 3) and anomalous diffraction data collected from GliJ crystals at the iron edge showed strong anomalous electron density for both active site metals (Figure 2C, upper left panel; Supplementary Figure 6A, B and Supplementary Table 1). The iron ions are coordinated by water molecules and surrounding amino acids. Notably, the residues engaged in metal binding are conserved between GliJ, HRD and Sco3058. The histidines 36, 214 and 235, Asp38 and Glu141 directly coordinate the metals, while H168 is hydrogen-bridged to the βiron via a water molecule (Figure 2C, upper left panel; amino acids are labeled according to the sequence alignment provided in Supplementary Figure 1). Asp38 undergoes two interactions with the α iron and is part of an unusual HxD (His-x-Asp, where x can be any amino acid) sequence signature, a motif actually typical of iron binding in most 223 oxoglutarate dependent oxygenases. Since many amidohy22 drolases instead feature an HxH motif, we converted the HxD sequence of GliJ to an HxH as well as an HxN feature and determined crystal structures of the respective GliJ D38H

RESULTS AND DISCUSSION Crystal structure of wild type (WT) GliJ. GliJ from A. fumigatus was expressed as a His6-tagged fusion protein in Escherichia coli, purified (Supplementary Figure 2) and crystallized. Diffraction data of WT GliJ crystals were collected to 2.1 Å resolution and the structure was phased by molecular 19 replacement using the coordinates of human HRD as a Patterson search model (Rfree 19.7%, PDB ID 5LWZ; Supplementary Table 1). Depending on the crystal space group, the asymmetric unit contains either one or three GliJ subunits of about 45 kDa, which in the crystal lattice form homodimers 2 (interface area ~ 3100 Å ). Each GliJ monomer adopts the triosephosphate isomerase fold, with a central barrel of βsheets surrounded by α-helices (Figure 2A). Its overall struc19 22 ture strongly resembles that of HRD and Sco3058 (rootmean-square deviation (r.m.s.d.) 0.7 Å, PyMOL; Supplementary Table 2). The C-terminus of GliJ, however, is helical and wraps around the second subunit (Figures 2A, B and Supplementary Figure 3). Although truncation of GliJ does not prevent dimer formation (Supplementary Figures 3 and 4A),

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Figure 2. Crystal structure of GliJ. (A) Ribbon and (B) surface representation of dimeric WT GliJ. The active sites in panel B are framed. (C) Zoom-in at the active site. The metal ions (large spheres) are coordinated by amino acids (labeled by the one-letter code) and water molecules (small spheres). GliJ can bind various metals, the anomalous electron densities of which are shown as green meshes contoured to at least 6σ. For calcium the FO-FC omit electron density map (yellow mesh, contoured to 5σ) is depicted. Despite the different ion radii, the protein side chains are rigid (Supplementary Figure 9K). However, the coordination sphere of the different metals is different. The upper left panel illustrates the originally determined GliJ crystal structure with iron bound at its active site. The nucleophilic water WN (purple sphere) is activated and coordinated by Asp304 (yellow). Asp38 (cyan) is part of the unusual HxD motif of GliJ. The other panels illustrate the results from metal exchange experiments.

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ions resulted in incorporation of the provided metals into the active sites with varying coordination spheres (Figures 2C and Supplementary Figure 9D–G, I–J). To test whether GliJ 2+ 2+ displays a metal preference, equal amounts of Zn , Mn , 2+ 3+ Fe and Fe were provided (each in 3.5 molar excess to the enzyme). According to X-ray crystallographic data and fluo2+ rescence spectra GliJ selectively grabs the Zn ions out of metal mixtures (Supplementary Figure 9H). From these data we conclude that GliJ can bind a large set of different metal 2+ ions, but that it prefers Zn .

(1.9 Å resolution, Rfree 19.0%, PDB ID 5LX4) and D38N mutants (1.95 Å resolution, Rfree 19.5%, PDB ID 5LX7; Supplementary Table 1 and Supplementary Figure 2). As proven by anomalous data collection, the D38H mutant (HxH) lacks the α iron and has the β iron bound only at low occupancy.His38 forms a strong hydrogen bond to Glu141 (2.8 Å) and hereby interferes with metal coordination (Supplementary Figures 6C and 7A, B). Mutation of Asp38 to Asn (HxN) also results in loss of the α iron, but high occupancy of the β iron is retained (Supplementary Figures 6D and 7A, C). While Asn38 does not participate in metal binding, a tris(hydroxymethyl)aminomethane (Tris) buffer molecule coordinates the β iron and complements its octahedral coordination sphere (Supplementary Figure 7C).

Using the GliK/GliJ coupled assay set-up, we found that iron-, zinc- and manganese-loaded GliJ preparations were active (Supplementary Figure 8B). These results are con14 sistent with previous observations and with the fact that certain hydrolytic metalloenzymes, e.g., glycerophos25 26 phodiesterases or aminopeptidases can accept distinct metal ions as Lewis acid catalysts.

Moreover, we mutated Asp304, that is, besides the metal ions, assumed to activate the nucleophilic water molecule for peptide bond hydrolysis. The effect of the D304A mutant on enzymatic activity was tested with a coupled GliK/GliJ assay, as the natural substrate of GliJ (GJS, Figure 1) is unstable and not accessible to isolation or chemical synthesis. Only upon the action of GliK that converts the bis-glutathione conjugate of the gliotoxin precursor into the substrate of GliJ, GliJ activity can be monitored by liquid chromatography–mass spec14 trometry (LC-MS). In agreement with the proposed function for Asp304, the catalytic activity of the D304A GliJ mutant is reduced (Supplementary Figure 8A) and the crystal structure lacks the nucleophilic water molecule (2.7 Å resolution, Rfree 22.0%, PDB ID 5LX1; Supplementary Table 1, Supplementary Figure 7A, D). Notably, corresponding mutations of Asp38 and Asp304 in Sco3058 were reported to render the 22 enzyme inactive.

Substrate binding. Next, we aimed to determine ligand complex structures of GliJ. Due to the inaccessibility of GliJ’s substrate, a Cys-Gly dipeptide was used for soaking and cocrystallization attempts but could not be trapped in the active site. Considering the large and complex nature of GliJ’s substrate, the dipeptide presumably was too small to be tightly bound in a defined orientation. Similarly, cocrystallization trials with cilastatin (CIL, Supplementary 27 Figure 10), an inhibitor of HRD, yielded only ligand-free structures. In agreement, cilastatin does not inhibit GliJ activity (Supplementary Figure 8A). Superposition of GliJ and the HRD:CIL complex structure excludes any steric reasons but indicates that the enlarged substrate binding pocket of GliJ might be insufficient for stabilizing the compound at the active site (Supplementary Figure 11).

Metal promiscuity. Considering that upon expression in 24 iron-rich LB medium, zinc-dependent proteins can be isolated with iron instead of zinc and taking into account that most metalloproteases, including GliJ homologues, are zincdependent, we investigated the metal preference of GliJ in more detail. First, we aimed at preparing metal-free enzyme. Denaturing and refolding GliJ in the presence of metal chelators yielded small amounts of native enzyme. Crystals of this preparation were devoid of any metals according to X-ray fluorescence scans (Supplementary Figure 9B). The 2FO-FC electron density map however clearly depicts one single ion 2+ at the active site, which most likely is Ca . According to its 2+ larger ion radius compared to iron, only one Ca ion fits into the active site of GliJ (Figure 2C, upper middle panel).

Hence, we explored inhibitor and substrate binding to GliJ by molecular modeling. To overcome the limitations of classical molecular docking approaches when dealing with chemically complex metal binding sites, we designed an original and elaborate strategy that combines molecular docking with subsequent molecular mechanics and mixed quantum mechanics/molecular mechanics (QM/MM) refinement calculations (Supplementary Methods; Supplementary Tables 4 and 5; Supplementary Text 1). Although modeling of CIL into iron- or zinc-bound GliJ yielded relevant conformations, their interaction energies were 50 to 60 kcal -1 mol less advantageous than those obtained for the HRD:CIL complex (Supplementary Table 4, Supplementary Figures 12 and 13). These theoretical data imply that CIL is less potent for GliJ than for HRD and corroborate our experimental data.

Since the yield of refolding was insufficient to conduct metal reconstitution experiments, subsequent crystallographic analyses and activity assays, the iron ions bound to native GliJ were removed by extensive treatment with the siderophore deferoxamine. Separation of iron-bound deferoxamine by size exclusion chromatography allowed for the addition of specific metal ions or buffer as a control either before or after crystallization. Finally, sophisticated anomalous X-ray data collection revealed the nature of the enzyme-bound metals (for details see experimental section). Using this approach, iron-free GliJ crystals were obtained. Although some zinc and copper ions were found to occupy the active sites in the control crystals (Supplementary Figure 2+ 2+ 3+ 2+ 2+ 2+ 9C), addition of >35 µM Zn , Fe , Fe , Mn , Ni or Co

To gain detailed insights into the binding mode of GliJ’s substrate (GJS, Figure 1) and the metal/ligand interactions at the catalytic active site, we performed molecular docking and QM/MM refinement calculations for the L-cysteinylglycinate (L-CG) moiety alone. Poses with favorable interaction ener-1 gies of about –30 kcal mol (Supplementary Table 4, Supplementary Figure 14) fulfill the coordination of both metal centers (either iron- or zinc-bound GliJ). While the Nterminus of L-CG is coordinated to the α metal center, one carboxylate oxygen and the amide oxygen of L-CG bind to the second (β) metal ion. The nucleophilic hydroxide is positioned between the two metals, 2.5–2.7 Å apart from the amide carbon atom of the substrate to be attacked (Supple-

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Figure 3. Modeling of the natural GliJ:substrate complex and the GliJ reaction intermediate. The best QM/MM refined poses for Ser Ser Ser GliJ’s substrate (GliJ:GJS conf. 3) and the GJSp intermediate (after processing of the L-CG moiety; GliJ:GJSp conf. 1) are presented as sticks bound to GliJ (gray surface; upper panels) in standard orientation. Amino acids from both GliJ chains (light and dark gray) are engaged in ligand binding. Polar and hydrophobic interactions of the substrate with the protein surroundings are shown as black dashed lines. The catalytic iron ions and the nucleophilic hydroxyl are shown as brown and purple spheres, respectively. Chemical structures of the modeled molecules are depicted in the lower panels. Note the tremendous rearrangeSer ment (rotation by 180°) and reverse binding mode of the intermediate GJSp with respect to GJS. 28

mentary Figure 15). Such a geometrical feature combined with the analysis of electronic descriptors (i.e. Mayer’s bond order for pairs of atoms directly involved in the reaction mechanism) is indicative of a potentially high reactivity of the related structures (Supplementary Table 4).

1, with iron and zinc in the active site, respectively; Supplementary Table 4). Since such a pose could not be identified during the docking of GJS, it seems to be favored only after Ser the cleavage of the L-CG side, thereby supporting the hypothesis of a sequential reactivity of the two substrate sides. Phe In agreement, processing of the L-CG residue prior to Ser cleavage of the L-CG moiety is disfavored (Supplementary Table 4 and Supplementary Figure 19). The fact that the nature of the active site metal does not significantly affect the modeling results in terms of energies, structures, and electronic features, conforms to the observed metal promiscuity of GliJ.

Next, calculations for the complete substrate, GJS, were performed. QM/MM interaction energies strongly favored an Ser orientation, in which the L-CG moiety occupies the active -1 site of GliJ (–27.26 and –30.87 kcal mol for GliJ:GJS conf. 3, with iron and zinc in the active site, respectively; Supplementary Table 4). The Ser side chain interacts with the protein backbone and the phenyl group is accommodated in a hydrophobic pocket (Figure 3 and Supplementary Figure 16). Furthermore, geometric and bond order descriptors showed a high propensity of this conformation to trigger the catalytic reaction (Supplementary Table 4). This conformation of GJS Ser suggests that GliJ at first cleaves the L-CG moiety and secPhe ondly processes the L-CG part (Figure 3). Docking of the Phe Phe two possible reaction products GJSp (processed L-CG ; Ser Ser Supplementary Figure 17) and GJSp (processed L-CG ; Supplementary Figure 18) yielded most favorable energies for Ser Phe the GJSp product with the L-CG part occupying the -1 Ser active site (–20.96 and –21.86 kcal mol for GliJ:GJSp conf.

Modelling of GliJ’s substrate GJS identified the P1’ site. Similar as in HRD and Sco3058, the P1’ pocket is open and spacious, suggesting that the P1’ residue is not stringently restricted to Gly. While HRD contributes to GSH decomposi29 tion by hydrolyzing L-CG dipeptides and GliJ removes Gly residues from GSH-conjugates, Sco3058 was shown to hydrolyze a broad range of dipeptide sequences with P1’ residues 22 larger than Gly. GliJ may also be promiscuous for the P1’ site, but gain specificity for GJS by interactions with the nonprimed pockets. This hypothesis is supported by the notion that GliJ residues predicted to interact with GJS (including

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Figure 4. Proposed reaction mechanism of GliJ. Upon deprotonation of a water molecule coordinated to Asp304 (step 1), the resulting nucleophilic hydroxide is placed in between the two metals (step 2). Processing of GJS, the substrate of GliJ, starts at Ser Ser the L-CG side chain (n° 1, blue pathway). After binding of the L-CG moiety the nucleophilic hydroxyl attacks the carbonyl Ser carbon of the Cys-Gly peptide bond (step 3.1). Upon cleavage of the scissile peptide bond (step 4.1), the GJSp intermediate is Ser released (step 5.1) and the catalytic site is ready for another hydrolysis of – either the L-CG moiety of a new molecule (n° 1, blue Phe Ser part) or the L-CG side chain of a partially processed GJSp fragment (n° 2, yellow pathway). Note: Besides divalent metal ions, GliJ is also able to bind trivalent ferric iron (Figure 2C). the C-terminus from the adjacent subunit) are not conserved in HRD and Sco3058.

tetrahedral intermediate that is stabilized by both metal ions (step 4.1). Upon breakdown of this intermediate and protonation of the amide nitrogen by aspartic acid 304, the first Ser cleavage product, glycine, is released (step 5.1). The GJSp product is displaced from the active site by water molecules that restore the catalytic center for another round of GJS hydrolysis or for the second cleavage of a semi-processed Ser GJSp molecule (back to step 1). The internal pseudosymmetry of the GJS molecule allows GliJ to remove both glycine Ser residues. After processing of GJS on the L-CG side, the Ser resulting intermediate GJSp can bind to GliJ in a reverse manner (rotation of ~85° along z and ~180° along y), thereby Phe positioning the L-CG moiety in the active site and enabling

Proposed reaction mechanism. Based on our structural data, the modeling results and previous reports on amidohy22, 30 we suggest the following reaction mechanism for drolases GliJ (Figure 4): Asp304 coordinates the nucleophilic water molecule (step 1) which is deprotonated with help of the βion and squeezed between the two metals (step 2). Binding Ser of the L-CG side of a GJS molecule to the active center displaces the water molecules engaged in metal coordination (step 3.1). Nucleophilic attack of the hydroxide onto the carbonyl carbon atom of the scissile peptide bond creates a

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ACS Chemical Biology per monomeric GliJ and an intramolecular disulfide bridge, the yields of native enzyme upon refolding were low.

the second cleavage (steps 3.2 to 5.2). Thus, GliJ requires affinity for two substrates – a feature that was also observed 31,32 for the S-methyltransferase of gliotoxin TmtA and that likely impedes the determination of well-defined ligand complex structures by X-ray crystallography.

Metal solutions. 10–100 mM stock solutions of metals were prepared in buffer D. All metals were used as sulfates, 2+ 2+ except for Ni and Co , which were applied as the respective chloride salts.

CONCLUSION

Metal exchange experiments. In order to test which metals can occupy GliJ’s active sites, the protein was purified by Ni-affinity and anion exchange chromatography and supplemented for three days with a final concentration of 10 mM deferoxamine. During incubation, the protein solution turned from colorless to pinkish to brownish, indicating the chelation of iron ions by deferoxamine. Size exclusion chromatography using buffer D served to separate GliJ from the chelator and yielded colorless protein. Upon dilution of the 2+ 3+ protein to 10 µM using buffer D, different metals (Fe , Fe , 2+ 2+ 2+ 3+ 2+ 2+ Zn , Mn ), metal combinations (Fe , Fe , Zn , Mn ) or buffer as control were added. The final concentration of each metal was at least 35 µM. In the case of mixtures, the metals were combined prior to the addition of the protein. After incubation over night at 4 °C, the protein-metal solutions were cleared by centrifugation. The supernatants were concentrated and individually applied to a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) using buffer D. Peak fractions showing the correct retention volume for native GliJ, were pooled, concentrated and subjected to crystallization.

The structural and mechanistic investigations on GliJ deliver profound insights into gliotoxin biosynthesis and ETP production in general. GliJ homologues are found in all ETP producers and are supposed to catalyze the same reaction although on distinct ETP backbones. Our X-ray data on a Cys-Gly dipeptidase involved in secondary metabolism now provide structural details on this type of enzyme also in comparison to similar proteins from other pathways, such as 33 the alternate GSH degradation in yeast or the detoxification of xenobiotics. Furthermore, we found that GliJ can incorporate numerous metals in its active site. While some metalloproteins strictly rely on one type of ion, certain hydrolytic 25, 26 34 enzymes, carboanhydrases , dioxygenases or superoxide 35 dismutases can accept different metals. Retaining their activity with distinct metals, these enzymes are termed cambialistic. Enzymatic activity however is not always a good criterion for determining the physiologically relevant ion, as in rare cases artificial metals can even booster enzymatic 36 activity and alter substrate specificity. EXPERIMENTAL SECTION

Activity assay. The enzyme assay was carried out as a cascade assay with GliK. Procedures for the expression and purification of the GliK enzyme as well as the isolation of the 14 GliK substrate were previously described. 80 µL of GliK -1 (1.5 mg mL in Tris/HCl pH 7.5; 150 mM NaCl), the respective -1 GliJ solution (final concentration 0.22 mg mL ) and 8 µL of -1 the GliK substrate (4 mg mL in H2O) were added to 150 µL Tris buffer (50 mM Tris/HCl, pH 7.5; 150 mM NaCl) and thoroughly mixed by pipetting. After incubation for 70 min at room temperature the reaction was quenched by addition of 200 µL of MeOH to precipitate the protein. Upon centrifugation (10 min, 13000 rpm) the supernatant was dried using a SpeedVac and reconstituted in 100 µL 50% (v/v) MeOH (H2O). LC-MS analysis was performed using an Exactive Orbitrap High Performance Benchtop LC-MS with an electrospray ion source (spray voltage 3.2 kV, capillary temperature 250 °C) coupled to an Accela HPLC system (Thermo Fisher Scientific, Bremen). HPLC conditions: C18 column (Betasil C18 3 µm 150 x 2.1 mm) and gradient elution (MeCN/0.1% (v/v) HCOOH (H2O) 5/95 for 1 min, going up to 98/2 in 15 min, then 98/2 for another 3 min; flow rate 0.2 mL -1 min ; injection volume: 10 µL). Compounds were identified by comparison with authentic references.

Purification of WT and mutant GliJ. WT and mutant GliJ cell pellets were resuspended in 100 mM Tris/HCl pH 7.5, 50 mM NaCl, 20 mM imidazole (buffer A) and lysed by sonification. Upon centrifugation at 21,000 rpm for 30 min at 4 °C, the supernatant was loaded on a 5 mL nickel chelating Sepharose FF column (GE Healthcare Life Science), pre-1 equilibrated with buffer A (flow rate 5 mL min ). After washing with buffer A, the protein was eluted by applying a 50 mL linear gradient from buffer A to buffer B (100 mM Tris/HCl pH 7.5, 50 mM NaCl, 500 mM imidazole). Subsequently, WT, D38H, D38N and G367* mutant GliJ were subjected to a 5 mL anion exchange chromatography column (YMC-BioPro Q30 -1 MiniChrom8-100; flow rate 1 mL min ) for further purification. While residual contaminants stuck on the anion exchange column pre-equilibrated with buffer C (100 mM Tris/HCl pH 7.5, 50 mM NaCl), GliJ proteins were found in the flow through. All GliJ proteins, including the N375* truncation mutant, were finally purified by size exclusion chromatography (Superdex 200, GE Healthcare) using buffer D (20 mM Tris/HCl pH 7.5, 100 mM NaCl). The GliJ mutant 14 D304A was purified as previously described for WT GliJ . Denaturation and refolding of GliJ. Upon Ni-affinity and anion exchange chromatography, GliJ was dialyzed against 4x 0.5 L 50 mM Tris/HCl pH 7.5, 50 mM EDTA, 8 M urea, 0.1% (v/v) β-mercaptoethanol and 1 mM deferoxamine over two days. By dialyzing four times against 0.5 L 250 mM Tris/HCl, pH 7.5, 400 mM arginine, 10% (v/v) glycerol, 0.5 mM reduced GSH and 1 mM oxidized GSH, the protein was stepwise refolded. Upon concentration the protein was diluted in a 1:1 ratio with buffer D and subjected to size exclusion chromatography. Due to the presence of 6 cysteines

Differential scanning fluorimetry. 1 μL of the fluorescent dye indicator SYPRO Orange (Sigma Aldrich, 5000 x in DMSO; 1:80 diluted in H2O) and 5 μg of purified WT and mutant GliJ protein were added per well in a 96-well thinwall PCR plate (nerbe plus). Buffer D (see gel filtration) was used to adjust the total volume per well to 20 µL. The plate was sealed and equilibrated to 25 °C for 10 min. Afterwards the samples were heated in a Stratagene Mx 3000P qPCR System (Agilent) from 25 to 95 °C in increments of 0.5 °C -1 min and the fluorescence was monitored using a CCD cam-

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era (Ex 492 nm/Em 610 nm). The melting point TM for each sample was determined by fitting the data (Boltzmann sigmoidal fit (least squares)) with GraphPad Prism 5. Data points before the minimum and after the maximum of fluo37 rescence intensity were excluded from fitting.

Present Address

Crystallization and structure determination of GliJ. Crystals of WT and mutant H6-tagged GliJ were grown by the sitting drop vapor diffusion technique at 20 °C. GliJ crystallized from 0.2 µL drops containing equal volumes of protein -1 (5–10 mg mL ) and reservoir solutions (0.1 M MES (2-(Nmorpholino)ethanesulfonic acid) pH 6.0, 5-30% (v/v) isopro2+ panol or 0.1 M MES pH 6.0, 10% (v/v) PEG6000). Co and 2+ Ni bound GliJ crystals were obtained by supplementing the mother liquor with a final concentration of 2 mM NiCl2 or CoCl2. All crystals were cryoprotected by the addition of a 1:1 (v/v) mixture of reservoir solution and 60% (v/v) glycerol and subsequently super-cooled in a stream of nitrogen gas at 100 K. Native and anomalous diffraction data were collected at the beamline X06SA, Swiss Light Source, Villigen, Switzerland. Fluorescence spectra served to identify the metals bound to GliJ. According to these spectra anomalous data sets were collected at the metal-specific absorption edges and, where necessary (e.g., upon crystal soaking), slightly above: zinc (λ = 1.281 Å), iron (λ = 1.738 Å), copper (λ = 1.378 Å), manganese (λ = 1.890 Å), nickel (λ = 1.484 Å), cobalt (λ = 1.604 Å). Reflection intensities were analyzed with the 38 program package XDS. Structure determination was per39 formed by Patterson search calculations with PHASER using the coordinates of the human renal dipeptidase (PDB 19 ID:1ITQ ). Cyclic refinement and model building steps were 40 41 performed with REFMAC5 and Coot. Water molecules 42 were placed with ARP/wARP solvent. Translation/libration/screw refinements finally yielded excellent values for Rcrys and Rfree as well as root-mean-square deviation (r.m.s.d.) bond and angle values. The models were prov43 en to fulfill the Ramachandran plot using PROCHECK 44 (Supplementary Table 1) and evaluated by MolProbity. Interface areas were calculated with PISA (‘Protein interfaces, surfaces and assemblies’ service PISA at the European 45 Bioinformatics Institute). Graphical illustrations were cre46 ated with PyMOL.

Funding Sources

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†present address: Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA Financial support by the Hans-Fischer-Gesellschaft (E.M.H.), the Peter und Traudl Engelhorn-Stiftung (E.M.H.), the Young Scholars Programme of the Bavarian Academy of Sciences and Humanities (E.M.H.) and the SFB749 (projects C08 (I.A.) and A10 (M.G.)) is acknowledged.

ACKNOWLEDGMENT We thank the TUM students S. Reisenauer, X. Mess and T. Zeitler for experimental and B. Urbansky (HKI) for technical support. We are grateful to the staff of the beamline X06SA at the Paul Scherrer Institute, SLS, Villigen, Switzerland, for assistance during data collection.

ABBREVIATIONS ETP, epipolythiodioxopiperazine; GJS, natural substrate of Ser Phe GliJ; GJSp , GJS processed at the serine side; GJSp , GJS processed at the phenylalanine side; GSH, glutathione; HRD, human renal dipeptidase; WT, wild type.

REFERENCES (1) Dagenais, T. R., and Keller, N. P. (2009) Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis, Clin. Microbiol. Rev. 22, 447–465. (2) Sugui, J. A., Pardo, J., Chang, Y. C., Zarember, K. A., Nardone, G., Galvez, E. M., Mullbacher, A., Gallin, J. I., Simon, M. M., and Kwon-Chung, K. J. (2007) Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone, Eukaryot. Cell 6, 1562– 1569. (3) Spikes, S., Xu, R., Nguyen, C. K., Chamilos, G., Kontoyiannis, D. P., Jacobson, R. H., Ejzykowicz, D. E., Chiang, L. Y., Filler, S. G., and May, G. S. (2008) Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence, J. Infect. Dis. 197, 479–486. (4) Scharf, D. H., Heinekamp, T., Remme, N., Hortschansky, P., Brakhage, A. A., and Hertweck, C. (2012) Biosynthesis and function of gliotoxin in Aspergillus fumigatus, Appl. Microbiol. Biotechnol. 93, 467–472. (5) Balibar, C. J., and Walsh, C. T. (2006) GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin, Biochemistry 45, 15029–15038. (6) Cramer, R. A., Jr., Gamcsik, M. P., Brooking, R. M., Najvar, L. K., Kirkpatrick, W. R., Patterson, T. F., Balibar, C. J., Graybill, J. R., Perfect, J. R., Abraham, S. N., and Steinbach, W. J. (2006) Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production, Eukaryot. Cell 5, 972– 980. (7) Kupfahl, C., Heinekamp, T., Geginat, G., Ruppert, T., Hartl, A., Hof, H., and Brakhage, A. A. (2006) Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model, Mol. Microbiol. 62, 292–302. (8) Gardiner, D. M., Waring, P., and Howlett, B. J. (2005) The epipolythiodioxopiperazine (ETP) class of fungal toxins: distri-

ASSOCIATED CONTENT Supporting Information Supplementary methods, text, figures, tables and references. This material is available free of charge via the Internet. Accession Codes: Coordinates and structure factors have been deposited in the RCSB Protein Data Bank under the accession codes 5LWZ (WT GliJ, space group C2), 5LX0 (WT GliJ, space group P3221), 5LX1 (GliJ-D304A), 5LX4 (GliJ-D38H), 5LX7 (GliJ2+ 2+ 2+ D38N), 5NRT (GliJ:Ca ), 5NRU (GliJ:Zn ), 5NRX (GliJ:Fe ), 3+ 2+ 2+ 2+ 5NRY (GliJ:Fe ), 5NRZ (GliJ:Mn ), 5NS5 (GliJ:Cu and Zn ), 2+ 2+ 5NS2 (GliJ:Co ), 5NS1 (GliJ:Ni ).

AUTHOR INFORMATION Corresponding Author *[email protected], *[email protected]

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Gliotoxin Biosynthesis: Structure, Mechanism, and Metal Promiscuity

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