Structure and Inhibition of the CO2-Sensing Carbonic Anhydrase Can2 from the Pathogenic Fungus Cryptococcus neoformans

doi:10.1016/j.jmb.2008.11.037

J. Mol. Biol. (2009) 385, 1207–1220

Available online at www.sciencedirect.com

Structure and Inhibition of the CO2-Sensing Carbonic Anhydrase Can2 from the Pathogenic Fungus Cryptococcus neoformans Christine Schlicker 1 , Rebecca A. Hall 2 , Daniela Vullo 3 , Sabine Middelhaufe 1 , Melanie Gertz 1 , Claudiu T. Supuran 3 , Fritz A. Mühlschlegel 2 and Clemens Steegborn 1 ⁎ 1

Department of Physiological Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany 2

Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK 3

Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy Received 8 August 2008; received in revised form 17 November 2008; accepted 19 November 2008 Available online 27 November 2008 Edited by G. Schulz

In the pathogenic fungus Cryptococcus neoformans, a CO2-sensing system is essential for survival in the natural environment (∼ 0.03% CO2) and mediates the switch to virulent growth in the human host (∼ 5% CO2). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the “plant-type” β-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors. © 2008 Elsevier Ltd. All rights reserved.

Keywords: β-class carbonic anhydrase; Cryptococcus neoformans; crystal structure; inhibition; sulfonamide

Introduction The ubiquitous gas carbon dioxide (CO2) is not only a by-product of cellular metabolism but also a nutrient and environmental signal.1 For example, CO2 levels control mammalian respiration,2 and CO2 is utilized in photosynthetic algae and plants as a nutrient and regulator.3 Recognition of CO2 is also involved in the regulation of virulence mechanisms of pathogenic microbes. For example, a decrease in CO2 induces gametogenesis of the *Corresponding author. E-mail address: [email protected]. Abbreviations used: CA, carbonic anhydrase; Cab, Methanobacterium thermoautotrophicum β-class carbonic anhydrase; ZBG, zinc binding group; AAZ, acetazolamide; PEG, polyethylene glycol; PDB, Protein Data Bank.

malaria pathogen Plasmodium,4,5 while increases in environmental CO2 trigger virulent growth of the pathogenic yeasts Cryptococcus neoformans and Candida albicans.6,7 C. neoformans is a ubiquitous human pathogen and causes life-threatening meningoencephalitis.8 The fungus lives in a natural environment with ∼ 0.03% CO2 but experiences a dramatic raise in CO2 concentration during transitions to its mammalian host (∼ 5% CO2). The increase in CO2 promotes biosynthesis of a polysaccharide capsule, which is an important virulence factor of C. neoformans.9 The CO2-sensing system of C. neoformans includes two prominent enzymes, the carbonic anhydrase (CA; EC 4.2.1.1) Can2 and the fungal adenylyl cyclase Cac1. Can2 catalyzes the formation of bicarbonate and a proton from CO2 and water. The bicarbonate activates the adenylyl cyclase Cac1, which promotes capsule biosynthesis.6,10 In an analogous manner, the CA Nce103 from another fungal patho-

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

1208 gen, C. albicans, forms part of a CO2-sensing system, triggering filamentous growth, which is a major virulence attribute.7 Fungal CAs including Can2

Crystal Structure of the Carbonic Anhydrase Can2

and Nce103 are essential for survival of C. neoformans serotype A in its natural environment and for C. albicans infections in epithelial virulence models.7

Fig. 1. Functional complementation of the C. albicans CA mutant with Can2 and alignment of Can2 with other β-CAs. (a) C. albicans strains RH1 (expressing the C. neoformans CA CAN2) and TK2a (expressing the native C. albicans CA NCE103), together with the CA null mutant control strains TK2 and TK2b, were streaked onto YNB agar and incubated in either 5.5% CO2 (left) or air (right) for 72 h. (b) Alignment of β-CAs with published structures from different species (sequence lines 3– 9; names given are PDB accession codes). The extended N-termini of the C. albicans (NCE) and C. neoformans (CAN2) isoforms are shown in red, and the three highly conserved, metal-binding amino acids are shown in blue. The conserved Asp/Arg pair is colored green, and the plant-type-specific amino acids are marked with orange boxes. The numbering corresponds to the Can2 sequence. For clarity, preceding N-terminal amino acids of 1ddz (1–27) and of 1ekj (1–37) are omitted. In the sequences of 1ym3 and 1ylk, N-terminal His tags are replaced by the original sequences. Amino acid positions are colored black, dark gray, and light gray, indicating decreasing conservation grades.

1209

Crystal Structure of the Carbonic Anhydrase Can2

Thus, they may constitute attractive targets for development of antifungals that target horizontal transmission and treatment of superficial skin infections. CAs are a large family of metalloenzymes well known for their “druggability”.11 The CA family is divided into five evolutionarily independent classes.12,13 The 16 known mammalian isoforms (I to XV) belong to the α-class14,15 and have been successfully used as targets for drug design and therapy,11 for example, for treatment of glaucoma with dorzolamide.16,17 Can2 and Nce103 belong to the β-class, which comprises a diverse set of CAs found in plants, algae, bacteria, and archaea.18 The γ-, δ-, and ζ-classes so far each consist of a single or few microbial enzymes.12,19–21 Although the CA families are unrelated in overall structure, the catalytic centers generally contain a zinc ion with tetrahedral coordination, except for the δ-CA CDCA1 (cadmium carbonic anhydrase 1), which instead contains a cadmium ion, and the γ-CA from Methanosarcina thermophila (Cam), which may employ zinc(II), cobalt(II), or iron (II).22 In α- and γ-class CAs, the zinc ion is surrounded by three conserved His residues, whereas in β-class CAs, it is coordinated by two conserved Cys residues and one conserved His residue.12 The fourth zinc coordination site is differently assigned: In some β-CAs, a water molecule or the product analog acetate was found at the fourth coordination site; in other β-CAs, the fourth position is occupied by a conserved aspartate.12 Both coordinations have been suggested to be part of the catalytic cycle; that is, the Asp might initially bind the zinc and gets replaced by the incoming water.23 The β-CAs can be further divided into two subclasses, the “plant-type” and the “Cab-type” class (named after the β-CA Cab from Methanobacterium thermoautotrophicum).24 The active-site residues Gln151, Phe179, and Tyr205 are conserved in plant-type β-CAs (denotation of the Pisum sativum CA24) but variable in the Cab-type β-CAs.12 Consistent with differences in active-site details, the two subfamilies show different susceptibility patterns for inhibition by known CA inhibitors.22,25,26 It thus appears that highly specific inhibitors can be developed, which even discriminate between β-class subfamilies and thus might be attractive leads for drug development. In order to reveal unique molecular features of the CO2-sensing CAs from fungal pathogens, we carried out a structural and functional characterization of Can2 from C. neoformans. The protein is functionally similar to Nce103 from C. albicans and we show that it can be inhibited potently by some of the known CA inhibitors. The crystal structure of Can2 shows that the enzyme belongs to the planttype β-CAs but carries a unique N-terminal extension, which can interact with the active-site entrance of the dimer. A structure of the Can2 complex with the inhibitor and product analog acetate reveals insights into interactions with physiological ligands and may lead to the design of even more potent inhibitors.

Results Can2 in vivo complementation In many organisms, including the pathogenic fungi C. neoformans and C. albicans, deletion of CA encoding genes is detrimental to growth in aerobic environments. To investigate whether Can2 from C. neoformans can functionally complement CA-deficient microorganisms—in particular whether it can substitute for the related CA Nce103 (see below)— we expressed CAN2 in an nce103 deletion mutant of C. albicans. CAN2 complemented the growth defect (although not as effective as the wild-type C. albicans NCE103 control) of the CA mutant, rescuing growth after 72 h (Fig. 1a). Furthermore, growth could be inhibited through supplementation of CA inhibitors (see below). The interesting observation that Can2 could only partially complement the growth defect of the C. albicans CA deletion strain is not thought to be due to insufficient CA activity, as Can2 has previously been shown to fully complement the growth of a CA-deficient Escherichia coli strain.10 Therefore, comparisons of the primary structures of Can2 and Nce103 with other β-CA isoforms were performed to identify potential differences. Both Nce103 and Can2 exhibit unusual N-terminal extensions (Fig. 1b) compared to other β-CAs. However, the N-termini are not highly homologous and Nce103 has an extension approximately twice as long as that of Can2. It is tempting to speculate that this protein region could be important to regulation of enzymatic activity (see below), and differences in these protein parts might provide some explanation for the partial complementation observed. Overall structure of Can2 In order to identify molecular details of Can2 that contribute to its specific function and might help in the development of specific inhibitors, we solved the crystal structure of the enzyme. Can2 crystallized in space group P3121 and its structure was solved by molecular replacement with an E. coli β-CA structure [Protein Data Bank (PDB) code: 1t75] as a search model (Fig. 2a). The structure was refined at 1.34 Å resolution to an Rcryst/Rfree of 13.8/18.5%. The asymmetric unit is composed of one Can2 monomer (containing residues 1–230 and an Nterminal PLGS sequence from the protease cleavage site, which is also visible in the electron density), one chloride ion, and one zinc ion. All residues are well ordered, except for the loop region encompassing residues 140–144. Two Can2 monomers form a tightly packed dimer exploiting the crystallographic 2-fold rotation axis, consistent with the dimeric behavior of Can2 observed in size-exclusion chromatography10 (also see below). The overall fold of Can2 (Fig. 2a and b) is similar to other β-CAs except for the N-terminal extension, which is longer than in other β-CA structures— except for the Porphyridium purpureum CA and the

1210

Crystal Structure of the Carbonic Anhydrase Can2

Fig. 2. Crystal structure of Can2. (a) Overall structure of the Can2 dimer. One monomer is colored blue, while the other one is colored cyan. The Zn2+ ions are shown as orange spheres. (b) Monomer of Can2. The monomer is shown in the same orientation as the dimer in (a). Secondary-structure elements are labeled and the positions of N- and C-terminus are indicated. (c) Comparison of Can2 with other β-class CAs. Depicted is an overlay of Can2 (blue) with the structures of the plant-type β-CAs of E. coli (yellow) and P. sativum (dark red) and the Cab-type β-CAs of M. thermoautotrophicum (green) and M. tuberculosis Rv1284 (gray).

P. sativum CA (Fig. 1b)—and sticks out of the bulk protein structure (see below). The N-terminal domain of the Can2 core is formed by four antiparallel αhelices (α1–4; Fig. 2b), which are packed against α5′ and β5′ of the second monomer within the dimer (Fig. 2a). The C-terminal domain contains a fivestranded β-sheet. Four parallel β-strands (β1–4) are shielded from solvent by six α-helices, and β5 formed by the Can2 C-terminus is attached antiparallel with β1–4. Between β3 and β4, four α-helices (α7–10) are inserted, and the complete C-terminal domain is packed between the N- and C-terminal domains of the partner monomer, resulting in a tight dimer interaction. Comparison to other β-class CAs The overall architecture of Can2 resembles the known β-class structures of the plant-type (P. sativum, PDB code: 1ekj; 24 P. purpureum, PDB code: 1ddz;23 E. coli, PDB codes: 1i6p 27 and 1t75; Haemophilus influenzae, PDB code: 2a8c;28 Mycobacterium tubercu-

losis Rv3588c, PDB codes: 1ym3 29 and 2a5v30 ) and Cab-type (M. thermoautotrophicum, PDB code: 1g5c;31 M. tuberculosis Rv1284, PDB code: 1ylk29 ) subclasses. Based on an overlay of the overall structures of Can2 and two representatives of each subclass, respectively (Fig. 2c), Can2 appears slightly closer related to the plant-type β-CAs from E. coli (r.m.s.d. 1.49 Å for 182 Cα atoms) and P. sativum (r.m.s.d. 1.55 Å for 166 Cα ) than to the Cab-type CAs from M. thermoautotrophicum (r.m.s.d. 1.70 Å for 112 Cα ) and M. tuberculosis Rv1284 (r.m.s.d. 1.69 Å for 129 Cα ). Major differences, however, are restricted to the N- and C-terminal regions. The N-terminus of Can2 has two additional α-helices and an extended structure sticking out of the protein structure (see below). The C-terminus in the Can2 structure is shorter than those of other plant-type β-CAs, more like the C-termini of the Cab-type structures from M. thermoautotrophicum and of M. tuberculosis Rv1284. It ends with β5 of the central β-sheet, without additional secondarystructure elements of plant-type CAs such as the

Crystal Structure of the Carbonic Anhydrase Can2

α-helices of the E. coli β-CA or the extended, additional β-strand of the β-CA from P. sativum. Can2 has a plant-type active site The active site of Can2 is located between β-strands β1/2 and α-helix α7. The catalytic zinc ion is coordinated by the side chains of Cys68, His124, and Cys127 (Fig. 3a). The fourth coordination site is

1211 occupied by a water molecule with an oxygen–metal distance of 2.07 Å. The position of the water molecule is stabilized through a hydrogen bond to the conserved residue Asp70, with a short distance of 2.62 Å, indicating a strong interaction. Asp70 is part of an Asp/Arg pair (Asp70/Arg72 in Can2) conserved in all β-CAs sequenced so far. The orientation of Asp70 is stabilized through interactions with the side chain as well as with the backbone of Arg72. The Asp/Arg

Fig. 3. Active site and inhibition of Can2. (a) Active site of Can2 with electron density contoured at 1.0 σ. Coordination of the active-site Zn2+ by Cys68, His124, Cys127, and a water molecule is indicated by black broken lines, and the hydrogen bond from the water molecule to Asp70 is colored orange. (b) Overlay of the active sites of Can2 and the plant-type β-CAs of E. coli (yellow) and P. sativum (dark red) and the Cab-type β-CAs of M. thermoautotrophicum (green) and M. tuberculosis Rv1284 (gray). The Zn2+ ions are shown as orange spheres, and the water molecules are colored according to the organism. (c) Chemical structure of the most potent inhibitors identified for Can2, AAZ, and benzolamide. (d) Detrimental effect of the Can2 inhibitors AAZ and benzolamide on the growth of the C. albicans Nce103 deletion mutant expressing CAN2 (RH1). RH1 was spotted onto YNB agar at 1 × 105, 1 × 104, and 1 × 103 cells/ml in the presence of (i) 4% DMSO and 3 mM benzolamide for 96 h or (ii) 4% DMSO and 3 mM AAZ for 72 h.

1212

Crystal Structure of the Carbonic Anhydrase Can2

pair has previously been proposed to be involved in proton shuffling12 and catalysis,24 which would be consistent with their conserved arrangement in Can2 and other β-CAs (Fig. 3b). Can2 comprises additional plant-type-specific conserved amino acids in its active site—Gln59, Phe87, and Tyr109 (corresponding to P. sativum Gln151, Phe179, and Tyr205, respectively), which clearly identify Can2 as a plant-type β-CA (Fig. 1b). These residues are in similar positions as in the P. sativum β-CA structure.24 Only Tyr109 (Tyr205 in P. sativum) is slightly shifted toward the active site in Can2, but this difference appears to be due to having a water molecule bound instead of the acetate molecule in the β-CA structure of P. sativum (see below). Inhibition of Can2 by sulfonamides and sulfamates Can2 possesses appreciable CO2 hydrase activity (kcat/Km of 4.3 × 107 M− 1 s− 1)32 similarly to other

CAs belonging to the α- or β-class. Further, Can2 and the human CA isoforms are inhibited appreciably by the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide; AAZ), with an inhibition constant of 10.5 against Can2. In order to identify compounds that show more distinct potencies against Can2, as opposed to human α-CAs, we tested various classical CA inhibitors of the sulfonamide/sulfamate type.11 We analyzed a panel of drugs in therapeutic use as αCA inhibitors (Table 1), such as AAZ and dorzolamide and a set of 22 simple sulfonamides widely used for the design of more potent CA inhibitors.11,33 Our data (Table 1) show that the sulfonamides/sulfamates inhibited Can2 appreciably, but their efficacy as inhibitors varied several orders of magnitude, with Ki values in the range of 10.5 nM to 32 μM. The highest potency was observed for AAZ (Ki = 10.5 nM) followed by benzolamide (Ki = 23 nM). Inhibitors also showing good potency (Ki b 100 nM) were aminobenzolamide (Table 1, compound 15), methazolamide, ethoxyzolamide, and brinzola-

Table 1. Can2 inhibition data with sulfonamides 1–22 and 15 clinically used derivatives AAZ–SAC Kia (nM) Inhibitor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 AAZ MZA EZA DCP DZA BRZ BZA TPM SLP IND ZNS CLX VLX SLT SAC

Compound name

hCA I

hCA IIb

Can2c

2-Amino-benzenesulfonamide (orthanilamide) 4-Amino-benzenesulfonamide (sulfanilamide) 4-(Carboxyethyl)-benzenesulfonamide 4-Methyl-benzenesulfonamide 4-Aminomethyl-benzenesulfonamide (homosulfanilamide) 4-Aminoethyl-benzenesulfonamide 3-Fluoro-4-amino-benzenesulfonamide 3-Chloro-4-amino-benzenesulfonamide 3-Bromo-4-amino-benzenesulfonamide 3-Iodo-4-amino-benzenesulfonamide 4-Amino-6-trifluoromethyl-benzene-1,3-disulfonamide 4-Amino-6-chloro-benzene-1,3-disulfonamide 5-Amino-1,3,4-thiadizole-2-sulfonamide 4-Methyl-5-imino-1,3,4-thiadiazoline-2-sulfonamide Aminobenzolamide 4-(4-Sulfanilyl-aminomethyl)-benzenesulfonamide 4-(4-Sulfanilyl-aminoethyl)-benzenesulfonamide 4-(2-Amino-pyrimidin-4-yl)-benzenesulfonamide Chlorazolamide 4-Hydroxymethyl-benzenesulfonamide 4-Hydroxyethyl-benzenesulfonamide 4-Carboxy-benzenesulfonamide Acetozolamide Methazolamide Ethoxzolamide Dichlorophenamide Dorzolamide Brinzolamide Benzolamide Topiramate Sulpiride Indisulam Zonisamide Celecoxib Valdecoxib Sulthiame Saccharin

45,400 25,000 6690 78,500 25,000 21,000 8300 9800 6500 6000 5800 8400 8600 9300 6 164 185 109 690 55 21,000 23,000 250 50 25 1200 50,000 45,000 15 250 12,000 31 56 50,000 54,000 374 18,540

295 240 495 320 170 160 60 110 40 70 63 75 60 19 2 46 50 33 12 80 125 133 12 14 8 38 9 3 9 10 40 15 35 21 43 9 5950

379 765 440 1150 18,490 1394 809 605 977 711 968 300 791 815 42 971 624 3887 379 623 878 484 10.5 63 87 1203 8347 87 23 367 812 963 971 3056 704 890 32,000

Data for hCA isoforms I and II are included for comparison from Refs. [11,33]. a Errors in the range of 5–10% of the data shown, from three different assays. b Human recombinant isozymes, stopped-flow CO2 hydrase assay method, pH 7.5. c Fungal recombinant enzyme, at 20 °C, pH8.3.

b

Crystal Structure of the Carbonic Anhydrase Can2

mide, which belong to rather different classes of sulfonamides, possessing various scaffolds. Generally, most of these compounds showed stronger inhibition of Can2 compared to hCA I but weaker Can2 inhibition when compared to hCA II,33 the two major cytosolic isoforms in humans and other vertebrates. The differences between the potencies against the different CAs indicate that development of highly specific inhibitors is possible, but the recognizable correlation between the effects on hCA II and Can2 indicates that further efforts will be needed for obtaining highly specific compounds, such as further variations on sulfonamides

1213 or the use of completely new compound classes (see below). Supplementing the medium with the two most potent Can2 inhibitors, AAZ and benzolamide, inhibited the growth of the C. albicans Nce103 deletion mutant expressing CAN2 (Fig. 3d). Growth was inhibited in low but not in high CO2 conditions. For therapeutic purposes, the concentrations of AAZ or benzolamide used (3 mM) would be too high. Concentrations needed for potent inhibition of the purified enzyme are much lower. This discrepancy may be attributed to the fungal cell wall, which constitutes a barrier for the inhibitor.

Fig. 4. Can2 complex with the product analog acetate. (a) Overall structure of the Can2/acetate complex (monomer C, gray) overlaid with the structure of uncomplexed Can2 (blue). The Zn2+ ions are shown as orange spheres. (b) Overlay of the active sites of the Can2/acetate complex and the Can2 structure with a bound water molecule (blue: Can2/water structure; aquamarine: Can2/acetate monomer A; cyan: Can2/acetate monomer B; gray: Can2/acetate monomer C). (c) Modeling of a Can2/benzolamide complex. Residues coordinating the active-site ion, restricting the space available to the inhibitor, or reachable for the inhibitor moieties are labeled.

1214 Therefore, screening for additional CA inhibitors should also involve their ability to transverse fungal capsules and cell walls to have maximal bioactivity. Can2 complex with the inhibitor and product analog acetate In order to rationalize the binding mode of sulfonamides such as AAZ, we tried to solve the crystal structure of a Can2 inhibitor complex. Soaking attempts, however, were not successful, likely due to blockage of the active site (see below). Cocrystallization resulted in a second Can2 structure in space group C2, which, surprisingly, had an acetate molecule bound to the zinc ion instead of the inhibitor in all three monomers of the asymmetric unit. Acetate was present at 200 mM in the crystallization drop. The fact that it was not replaced by 4 mM of the tight binding compound benzolamide (Ki = 23 nM) could indicate that acetate might be a good binding ligand for Can2. Indeed, testing acetate revealed that it inhibits Can2 with micromolar affinity (Ki = 10 μM). This finding indicates that carboxylate-based inhibitors might be useful for the development of potent, specific inhibitors for Can2 and possibly for related β-CAs, which will be a topic for our subsequent studies. The Can2/acetate complex shows the interactions of the enzyme with a surprisingly good “zinc binding group” (ZGB; of the carboxylate type) as well as with an analog of the reaction product, that is, bicarbonate. The overall structure of the Can2/ acetate complex is very similar to that of the Can2 structure without ligand (Fig. 4a), except for the position of the N-terminal extension (see below). The asymmetric unit contains one glycerol molecule and three Can2 monomers (monomer A: completely defined from Pro2 to Phe231; monomers B and C: flexible loops 141–143 and 140–142, respectively, are missing), each harboring one zinc ion and one acetate molecule. The acetate fills the fourth coordination site of the catalytic zinc ion by binding with one of its oxygen atoms (Fig. 4b) with a distance between 1.89 and 2.15 Å. The acetate molecule is located close to OD2 of Asp70 (distances between 2.70 and 3.41 Å) and in the neighborhood of the NH2 group of Arg72 (the shortest distance is observed in monomer A, acetate position A with 3.81 Å, others are 5 Å and longer), and its carboxyl group forms hydrogen bonds to NE2 of His124 (distances, 2.95–3.50 Å) and the backbone of Gly128 (distances, 2.84–3.27 Å). The

Crystal Structure of the Carbonic Anhydrase Can2

methyl group of the acetate is oriented toward Val92. There are, however, small differences in the acetate binding details between the monomers. In monomer A, the acetate has two orientations occupied half–half. It is tilted toward Arg72 in orientation B compared to orientation A, which resembles those found in the other monomers. The acetate carboxyl group appears to form an additional, weak interaction to NE2 of Gln59 with different O–N distances (3.70 Å in position A, monomer A; 3.37 Å in position B, monomer A; 3.69 Å in monomer B; 3.1 Å in monomer C). Despite the slightly different orientations of the acetate molecules, the positions of active-site residues in the different Can2 monomers as well as in the Can2 structure without ligand stay nearly the same. Some of the interacting residues are shifted about 0.5–0.7 Å toward the acetate molecule, whereas the conserved Tyr109 appears to slightly shift away from the ligand to provide space. In order to rationalize the interactions of bulkier inhibitors of the sulfonamide family with Can2, we manually placed benzolamide with its sulfonamide onto the acetate of the Can2/acetate structure, with the NH2 group of the sulfonamide binding to the zinc ion (Fig. 4c). Orienting the remaining part of the inhibitor into the only large pocket available, the channel from the active site toward the solvent, results in a severe clash with the N-terminal “lid” closing this channel, but we assume that this lid can be reoriented in order to allow open access to the active site (see below). After removing the Nterminal lid, the bottleneck for inhibitor binding is formed by the side chain of Tyr109 and by the backbone of Gly128 and Gly129. These amino acids have to shift away to enlarge the active-site area on top of the Zn2+ ion to accommodate the inhibitor. In contrast, the conserved Asp70/Arg72 pair is already in a favorable position, possibly supporting inhibitor binding through hydrogen bond formation of the Arg72 side chain. The terminal, phenyl moiety of the inhibitor is located between the end of α6′ (Asn113 and Val114) and the loop between α4 and β1 (Gly58 and Gln59). Rearrangements of these residues, which provide favorable hydrophobic interactions for binding, might occur. Interestingly, the Tyr109 and Gln59 residues conserved in the plant-type subclass of the β-CAs appear to be involved in inhibitor accommodation, which might explain the previously observed differences in inhibitor sensitivity between the two β-CA subtypes.12

Fig. 5. The unique N-terminus of Can2 and related CAs. (a) Two symmetry-related dimers interact through the N-terminal extension of Can2. One dimer is shown in surface representation (gray), showing the crevice that accommodates the extended N-terminus of the second dimer shown as cartoon (blue and cyan). The N-terminal residues (amino acids − 3 to 10) laying in the crevice are shown in stick representation. (b) Closer view of the interaction between the N-terminal extension (amino acids −3 to 10) and the electrostatic surface of the dimer. This figure was prepared using GRASP2.34 (c) Interactions of the N-terminal extension with the binding crevice of the same dimer observed in the structure of the acetate complex. For clarity, the N-terminus of monomer B (gray) is colored yellow. Hydrogen bonds are marked with black broken lines. (d) Model of a Can2/benzolamide complex, showing the steric clash between the inhibitor and the N-terminal extension covering the active-site entrance (green). (e) Models for potential functions of the N-terminal extension of Can2.

Crystal Structure of the Carbonic Anhydrase Can2

Fig. 5 (legend on previous page)

1215

1216 The N-terminus of Can2 forms an extended tail In contrast to many other β-class CAs, Can2 and Nce103 of the fungal CO2-sensing systems have Nterminal sequence extensions of about 26 and 63 residues, respectively. Only the N-termini of the P. purpureum and the P. sativum CAs display a similar feature, but their extensions are not obviously related in sequence to the fungal enzymes (Fig. 1b). However, the crystal structure of the P. sativum CA does not include the extended N-terminus. In the P. purpureum CA, the N-terminus forms an additional α-helix (α1), which packs against an α-helix (α18) and β-strand (β10), thereby tightening the dimer. In Can2, these residues form two additional α-helices and an extended structure sticking out from the body of the dimer (Fig. 2a). The extension is oriented toward a symmetry-related dimer, where its interaction with a surface groove covers approximately 600 Å2 (Fig. 5a and b). The groove area is located on top of the active site and formed by residues of α7, α9, and β3–5 and also comprises residues of α4′ and α6′ of the second monomer within the dimer. The interaction has a strong hydrophobic component and also features several hydrogen bonds (Fig. 5b and c) formed by Phe3, Ala5, and Lys9 from the Nterminal extension to Val56, Arg72, Asp168, and Lys174 of the binding groove. Interactions are formed by Ala5 (backbone NH) to Val56 (carboxyl oxygen; distances between 2.80 and 3.13 Å) and Lys9 (NZ) to Asp168 (OD2; distance, 2.93 Å, only in the uncomplexed Can2 structure). Furthermore, there is an interaction between Asp12 (OD1) and Lys174 (NZ; distances between 2.99 and 3.34 Å). Interestingly, the side chain of Arg72 (NH1), the conserved residue that orients the active-site residue Asp70, forms a hydrogen bond to the carboxyl oxygen of Phe3 (distances between 2.82 and 3.22 Å) in all monomers except for monomer C of the acetate complex where Phe3 is not visible in the electron density. The N-terminal extension of the Can2-like CAs thus not only covers the active-site entrance but also directly interacts with a conserved active-site residue. In both Can2 structures, the N-terminal extension is oriented toward a symmetry-related dimer, except for monomer B in the acetate complex, where the extension is folded back around Leu18 and packed against the same dimer (monomers B and C) into the interaction groove on top of the active site of monomer C. In this interaction mode, the Pro7/ Asn170 interaction appears to be weaker (with a distance of 3.8 Å compared to 3.1–3.2 Å with extended N-terminus). In the Cab-type M. thermoautotrophicum β-CA, the N-terminus is also positioned outside of the core structure and packed against another dimer, but its N-terminal helix is much shorter than the Can2 extension and located near β5 instead of the surface groove on top of the active site. In other β-CAs, there are various oligomerization states: dimer (E. coli), tetramer (H. influenzae), and octamer (P. sativum), but none exploits an interaction resembling the one observed in the Can2 crystals. The tetramer of H.

Crystal Structure of the Carbonic Anhydrase Can2

influenzae is formed by two dimers packed against each other via the surface of α-helices α4, α6, α7, and α8. The octamer of P. sativum is formed via the Cterminal β-strand, which binds to another dimer. Additional size-exclusion chromatography experiments with Can2 in a buffer similar to the crystallization condition [buffer containing 5% polyethylene glycol (PEG) 3350] revealed, in addition to the dimer, higher-order oligomers corresponding to a tetramer and a hexamer or octamer. Considering the fact that each dimer interacts in the crystal via its two N-termini with two dimers widely separated in space, leading to an almost infinite crystal packing network, indicates that this interaction mode is likely a crystallization artifact, apparently induced through the crystallization buffer. We thus assume that under physiological conditions, either the interdimer interaction mode is used slightly differently so that defined oligomers (tetramer, octamer) are assembled or the interaction mode of the N-terminus within the dimer might be physiologically relevant. Irrespective of this question, the interaction of the N-terminus with active-site residues, especially Arg72, might influence the enzyme's activity, and the active-site entrance conformation without interacting N-terminus could constitute a different activation state (activated or inhibited). Interestingly, binding of inhibitors such as benzolamide requires a dramatic reorientation of the Nterminus in order to provide the necessary space (Fig. 5d). This finding (a) explains the difficulty to obtain a crystal structure of a Can2/inhibitor complex and (b) might indicate that understanding the action of the Nterminus should help in the development of more specific inhibitors due to their overlapping sites of action. Furthermore, a BLAST search with the sequence of the N-terminal extension revealed no homologs other than fungal CAs. We thus speculate that the N-terminus mediates a regulation mechanism or protein/protein interaction specific for this family of fungal enzymes (Fig. 5e), a feature that might be helpful for specifically targeting these proteins by therapeutic agents.

Discussion The CA enzyme family is well known and long used as therapeutic target, for example, for developing treatments of glaucoma (hCA XII) and tumors (hCA IX and XII).11 These efforts have so far been focused on the human CAs, which belong to the α-subfamily. The fact that Can2 and the related Nce103 are essential for aerobic growth and their role in regulating fungal pathogenesis6,7,10 suggests that these enzymes could make good targets for antifungals. Can2 and Nce103 belong to the β-CA class, which has no mammalian members, indicating major differences from the host CAs, which should enable highly specific inhibition for antifungal development and physiological studies on fungal pathogenesis. We thus identified and described here several unique features of the Can2-like β-class CAs of the fungal CO2-sensing systems.

1217

Crystal Structure of the Carbonic Anhydrase Can2

Can2 is an enzyme with appreciable activity for the hydration of carbon dioxide to bicarbonate and protons, comparable to several α-CA isozymes.35 The activity of this enzyme is also inhibited by compounds of the sulfonamide/sulfamate family widely used as CA inhibitors. The efficient inhibition with AAZ, a compound of more than 20-fold lower potency against hCA I (Table 1),11 indicates that it should be possible to develop these compounds into specific inhibitors. The comparable affinity of AAZ, and other potent Can2 inhibitors, against hCA II shows that other chemical moieties fused to the sulfonamide ZBG would have to be explored for this purpose. Considering that the potencies of dorzolamide and sulthiame for hCA II are increased by 2–3 orders of magnitude compared to Can2 provides strong evidence that such variations can distinguish specific features of these CA enzymes. The structure of Can2 identifies two interaction environments that should be considered in the design of novel compounds. First, longer compounds, such as benzolamide, appear to exploit the pocket between α6′ and the loop between α4 and β1, which includes the Gln59 conserved in plant-type β-CAs. α-CAs generally have a deeper, differently shaped active-site cleft, and exploiting this pocket thus is highly attractive for improving inhibitor specificity. Second, the “bottleneck” region including the plant-type characteristic Tyr109 restricts access to the active site and thus is in close contact with compounds targeting the catalytic zinc ion; in fact, rearrangements of these residues have to occur in order to accommodate organic compounds such as the potent Can2 inhibitor AAZ. This finding is an important difference to hCA I, which generally shows a low affinity for this class of inhibitors compared to hCA II and Can2, due to its rather restricted active-site cavity36 that appears to be unable to adjust to larger ligands in the way Can2 does. Further structural studies on Can2/inhibitor complexes will be helpful to better understand these differences and for fully exploiting the identified Can2 interaction areas. When trying to crystallize a Can2/inhibitor complex, we surprisingly found that the enzyme's active site contained an acetate molecule from the crystallization solution as zinc ligand. Similarly, the product mimic acetate contained in the crystallization solution was found in the active site of the β-CAs from P. sativum,24 whereas the active sites of an M. thermoautotrophicum β-CA 31 and M. tuberculosis Rv128429 contained the CA substrate water instead. In a third group of β-CAs, those from P. pupureum,23 E. coli,27 H. influenzae,28 and Rv3588c from M. tuberculosis,29 the fourth position is occupied by the conserved Asp70 (Can2 numbering). All three ligands are likely to represent states of the catalytic cycle: The aspartate ligand would be the initial and end state, the water ligand is a substrate complex, and the acetate ligand mimics a product complex. However, the efficient coordination of the zinc by the carboxyl groups of aspartate and acetate indicates that the active-site environment of β-CAs favors such a zinc/carboxylate interaction. Consis-

tently, we found that acetate has a low micromolar affinity for Can2. Acetate shows lower affinities to α-CAs,37 and the carboxyl group thus is a ZBG with little relevance for the design of α-CA inhibitors.11 Our results suggest it instead as an attractive ZBG for the development of compounds that specifically inhibit microbial β-CAs. A major difference to other CAs, even within the β-class, is the N-terminal extension of Can2. No significant structural similarity to known protein structures deposited with the PDB was found for the N-terminus, but our structure shows that this protein part can interact with a channel crossing the active-site entrance. We find this N-terminus/ channel interaction either within a dimer or as a cross-link between two dimers. The higher-order oligomerization through the cross-linking interaction appears to be a crystal packing interaction rather than a physiologically relevant interaction (see above). We assume that the N-terminus is an internal regulator or an interaction site for a different protein (Fig. 5e). In fact, both possibilities might be true and a regulatory protein could compete with the Can2 channel for binding of the Nterminus and thereby influence Can2 activity. The positions of the N-terminus differ by up to 5–6 Å between individual monomers in the region around residue 18, which indicates that this area acts as a hinge and enables a dynamic change of the orientation of the N-terminus. The efficient inhibition of Can2 by benzolamide also indicates that a different orientation of the N-terminus is possible, as it clashes with the inhibitor when bound to the Can2 channel. We thus speculate that the Nterminus acts as a switch and it will be interesting to see which physiological factor, such as pH, a small molecule, or a protein, might trigger this switch. However, binding to another protein could also simply have the function to specifically localize Can2, and future research will have to reveal the role of this protein part.

Materials and Methods Cloning, protein expression, and purification Full-length Can2 (SWISSPROT code: Q3I4V7) was cloned into the pGEX6P2 expression vector with an N-terminal glutathione S-transferase tag. Can2 protein was expressed in E. coli BL21(DE3) for 18 h at 20 °C after induction with 0.5 mM IPTG. Harvested cells were disrupted in a French Press and cell debris was removed by centrifugation at 4 °C at 18,000 rpm for 45 min in an HFA 22.50 rotor. The protein was applied on a glutathione S-transferase Sepharose column (GE Healthcare) and incubated with PreScission Protease overnight at 4 °C. Cleaved protein was washed from the column with buffer 1 (150 mM NaCl, 50 mM Tris–HCl, pH7, 1 mM ethylenediaminetetraacetic acid, and 1 mM DTT) and concentrated in a Centricon 10 device. Subsequently, Can2 was subjected to size-exclusion chromatography in buffer 2 (20 mM Tris, pH 7.8, and 25 mM NaCl). Chromatography fractions were analyzed by SDS-

1218

Crystal Structure of the Carbonic Anhydrase Can2

PAGE, pooled, concentrated in a Centricon 10, frozen in aliquots in liquid nitrogen, and stored at −80 °C. In vivo complementation Full-length CAN2 was cloned into the C. albicans integrative pFM2 vector, under the control of the Tef2 promoter, utilizing the PstI and BamHI restriction sites. The resulting vector (pRH1) was linearized with NheI and transformed into the C. albicans CA mutant (nce103/ nce103), TK1 (see Table 2 for strains), at the URA3 locus as described previously.7 Transformants were selected on YNB agar (2% glucose, 1 × Difco YNB without amino acids, and 2% agar) in an atmosphere of 5.5% CO2. The resulting strain, RH1, was streaked in parallel with the C. albicans control strains TK2, TK2a, and TK2b. Growth was observed at 37 °C in air and 5.5% CO2 for 72 h. For the in vivo CA inhibition, RH1 was spotted (5 μl) onto YNB agar at concentrations of 1 × 105, 1 × 104, and 1 × 103 cells/ml. AAZ and benzolamide were dissolved in 100% dimethyl sulfoxide (DMSO) and supplemented into YNB agar at a concentration of 3 mM with a final concentration of 4% DMSO and incubated for 72 and 96 h, respectively.

Table 3. Data collection and refinement statistics

Space group Unit cell constants a (Å) b (Å) c (Å) α (°) β (°) γ (°) Resolution (Å) Unique reflections 〈I/σ〉 Completenessa (%) Rmergea (%) Refinement resolution (Å) Reflections used for refinement Protein atoms Ligand atoms Solvent atoms r.m.s.d. bond lengths (Å) r.m.s.d. bond angles (°) Average B-factor (Å2) Final Rcryst/Rfreeb (%)

Can2

Can2 acetate complex

P3121

C2

55.19 55.19 134.59 90 90 120 47.8–1.34 54,533 15.43 (2.67) 99.8 (97.8) 7.0 (21.4) 47.8–1.34 54,510

177.98 56.59 83.22 90 116.58 90 46.8–2.05 46,398 13.18 (3.01) 98.5 (97.1) 12.9 (49.1) 46.8–2.05 46,398

1935 2 165 0.01 0.03 17.3 13.8/18.5

5432 21 144 0.02 1.5 34.1 18.7/23.1

a

CA activity and inhibition assay

Numbers in parentheses are for the outermost shell. Rfree was calculated from 5% of measured reflections omitted from refinement. b

An Applied Photophysics stopped-flow instrument was used for assaying the CA-catalyzed CO2 hydration activity.38 Phenol red (0.2 mM) was used as indicator, measured at 557 nm, with 10 mM Hepes (pH 7.5) or Tris (pH 8.3) as buffer and 20 mM Na2SO4 or 20 mM NaCl (for keeping the ionic strength constant). Measurements were done for a period of 10–100 s. CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were used for determining initial velocities. Noncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 mM) were prepared in distilled, deionized water with 10–20% (v/v) DMSO (which is not inhibitory at these concentrations) and dilutions up to 0.01 nM with distilled, deionized water. Enzyme solutions were preincubated with inhibitors for 15 min at room temperature prior to the assay. Inhibition constants were obtained by nonlinear least-squares methods using PRISM 3, from Lineweaver– Burk plots, as reported earlier,38 and represent the mean from at least three different determinations. Crystallization, data collection, and structure determination Can2 was crystallized without ligand at 293 K by mixing 1 μl protein solution (17 mg/ml) and 1 μl reservoir Table 2. C. albicans strains used in this study Strain TK1 TK2 TK2a TK2b RH1

Genotype

Reference

nce103Δ∷hisG/nce103Δ∷hisG ura3∷imm434/ [7] ura3∷imm434 nce103Δ∷hisG/nce103Δ∷hisGura3∷imm434/ [7] ura3∷imm434-(URA3) nce103Δ∷hisG/nce103Δ∷hisGura3∷imm434/ [7] ura3∷imm434-(NCE103) nce103Δ∷hisG/nce103Δ∷hisG ura3∷imm434/ [7] ura3∷imm434-(pSM2) nce103Δ∷hisG/nce103Δ∷hisG ura3∷imm434/ This report ura3∷imm434-(pRH1)

(20% PEG 3350, 0.2 M NaCl, and 0.1 M Tris–HCl, pH 8.6). Crystals grew within 1 day. Crystals were transferred to cryosolution containing the reservoir components plus 15% 2,3-butandiol as cryoprotectant. For crystallization of Can2 with acetate, 1 μl protein solution (17 mg/ml) was mixed with 1 μl reservoir solution (24% PEG 8000, 0.2 M ammonium acetate, 0.01 M magnesium acetate, 0.05 M sodium cacodylate, pH 6.6, and 250 μM benzolamide) and microseeded after 1 day with Can2 crystals without acetate. Crystals were obtained within 1 week, soaked in 4 mM benzolamide overnight, and then transferred to cryosolution containing the reservoir components plus 10% glycerol. Diffraction data sets were collected at 100 K at Swiss Light Source beamline X10SA. Indexing, scaling, and merging were done with XDS39 (Table 3). The structure of Can2 in space group P3121 was solved by molecular replacement with PHASER40 using the β-CA from E. coli (PDB code: 1t75, residues 2–215) as a search model. The initial model was improved by using automatic model tracing of ARP/wARP.41 For completing the model, manual building with Coot42 was alternated with refinement with REFMAC.43 Anisotropic refinement and H-atom placing were included in the final steps of refinement, which were done by using SHELXL.44 For solving the structure of the Can2/acetate complex crystallized in space group C2, residues 11–230 of Can2 in P3121 were used as search model for molecular replacement using PHASER. After rigid-body refinement using REFMAC, the model was improved by rebuilding in Coot and refinement in REFMAC. The PRODRG server45 was used for building coordinate and parameter files for acetate. The structures were analyzed by using Coot and SFCHECK,46 and structural figures were generated with PyMOL† if not stated otherwise.

† http://pymol.sourceforge.net

Crystal Structure of the Carbonic Anhydrase Can2 Coordinates Coordinates and structure factors for the crystal structure of Can2 and the Can2/acetate complex have been deposited with the PDB under accession codes 2w3q (Can2) and 2w3n (Can2/acetate).

Acknowledgements We greatly acknowledge technical assistance from Barbara Kachholz. We thank the beamline staff of X10SA at the Swiss Light Source Paul Scherrer Institute, Villigen, CH, for support and our colleagues from the Max-Planck-Institute for Molecular Physiology, Dortmund (Dept. Wittinghofer and Dept. Goody) for help with data collection. This work was supported by Deutsche Forschungsgemeinschaft grant STE1701/2 (to C.S.). Work in the Mühlschlegel laboratory is funded by the Medical Research Council, and work in the Supuran laboratory is funded by the European Union (project DeZnIT).

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