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Crystal analysis of aromatic sulfonamide binding to native and (Zn)2 adduct of human carbonic anhydrase I Michigan 1
Inorganica Chimica Acta 339 (2002) 135 /144 www.elsevier.com/locate/ica
Crystal analysis of aromatic sulfonamide binding to native and (Zn)2 adduct of human carbonic anhydrase I Michigan 1 Marta Ferraroni a, Fabrizio Briganti a, W. Richard Chegwidden b, Claudiu T. Supuran a, Andrea Scozzafava a,* a
Laboratorio di Chimica Bioinorganica, Dipartimento di Chimica, Universita` degli Studi di Firenze, Via della Lastruccia, 3, I-50019 Sesto Fiorentino, Italy b Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, USA Received 4 November 2001; accepted 30 January 2002 Dedicated in honor of Professor Helmut Sigel
Abstract The crystal structures of sulfanilamide (4-aminobenzene-sulfonamide) complexed to the enzyme variant human carbonic ˚ resolution respectively are described. anhydrase I Michigan 1 (CA I M1) and to its (Zn)2 adduct refined at 2.2 and 2.6 A Comparisons among these structures and the corresponding sulfonamide adduct of human CA I show significant differences in the orientation of the inhibitor molecule and in its interactions with active site residues such as His200, Thr199, Leu198, Gln92, and Arg/His67 which are known to play important roles in substrate or inhibitor recognition and binding. In CA I M1 molecule B a lengthening of the Zn/N1 sulfanilamide bond distance and a corresponding shortening of the distance between the sulfonamido group and Thr199 are observed compared with the values for native CA I. When the second Zn(II) ion is present in the active site, the p -amino group and the aromatic ring of the inhibitor molecule appear to tilt towards Gln92 and Arg67, moving away from residues His200 and Leu198. The structural differences in inhibitor binding between the CA I isozyme and the CA I M1 variant are discussed in terms of the different inhibition constants measured for a variety of aromatic and heterocyclic sulfonamides. # 2002 Elsevier Science B.V. All rights reserved. Keywords: X-ray crystallography; Zinc enzymes; Carbonic anhydrase; Genetic variant; Point mutation CA I Michigan 1; Sulfonamides; Sulfanilamide; Inhibition
1. Introduction Carbonic anhydrase isoenzymes (CA, EC 4.2.1.1) are seemingly ubiquitous, well characterized metalloenzymes that are known to occur in three gene families a, b and g. The human isozymes all belong to the afamily and are monomeric, containing one zinc ion per polypeptide chain (Mr /30 kDa) [1]. These highly abundant proteins are involved in critical physiological processes connected with respiration and transport of CO2/HCO3 between metabolizing tissues and the lungs, pH homeostasis, electrolyte Abbreviations: CA, Carbonic anhydrase; HCA, Human carbonic anhydrase; CA I M1, Carbonic anhydrase I Michigan 1. * Corresponding author. Fax: /39-055-457 3385 E-mail address: [email protected] (A. Scozzafava).
secretion in a variety of tissues/organs, and biosynthetic reactions, such as gluconeogenesis, lipogenesis and ureagenesis [1,2]. The X-ray structures of some isozymes have shown that the zinc ion is coordinated to three histidines and a water molecule/hydroxide ion [3 /7]. The catalytic mechanism of CAs has been extensively investigated through kinetic, spectroscopic and structural studies [8 /10]. The enzyme can also act on other carbonyl systems, such as esters and aldehydes [11]. Catalysis of both ester hydrolysis and aldehyde hydration appears to occur by the zinc-hydroxide mechanism as for the CO2 hydration reaction [10]. Inhibition of several of these enzymes by aromatic/ heterocyclic sulfonamides has been exploited clinically for more than 45 years in the treatment of a variety of
0020-1693/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 0 2 ) 0 0 9 5 9 - 3
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diseases such as glaucoma, epilepsy, congestive heart failure, mountain sickness, gastric and duodenal ulcers [12 /16]. The inhibitory mechanism is primarily controlled by the coordination of the sulfonamide group to the catalytic zinc ion and secondarily by finely tuned tight binding to other regions of the active site cavity [17 /25]. A variety of CA activators has also been recently investigated and the structural characterization of some of their adducts with CA II has led to the identification of a novel ‘activator binding site’ in the CA cavity [26,27]. Many mutations of active-site residues of human carbonic anhydrase (HCA) II resulting in changes of CO2 hydration as well as of esterase activity and inhibitor-binding properties have been extensively studied, permitting identification of the amino acid residues that play important roles in catalysis. In CA I, which possesses about one fifth of the activity of CA II, the situation is less clear; the pKa of His 64 is too low for efficient proton transfer and the specific pathway or pathways of protons involving shuttle groups remain to be established [10,28]. Several active-site mutants of CA I have been prepared by site-directed mutagenesis in order to try to understand the striking catalytic differences between CA I and CA II, the two isozymes that predominate in the red cell [29]. A number of naturally occurring variants of mammalian CA isozymes have also been reported [30]. One variant of human CA I (CA I Michigan 1) has attracted particular interest, since it provides an example of a point mutation that results in true metal ion activation of the gene product [31]. Although its CO2 hydration and esterase activities appeared to be within the normal range, its esterase activity toward a- or b-naphthyl acetate was enhanced tenfold at a Zn(II) concentration of 10 4 M [32,33]. The elusive mutation conferring the above reported properties was recently revealed by Chegwidden et al. and turned out to be His67Arg [34]. Other studies from our laboratory have also revealed that, although CA I Michigan 1 (CA I M1) was inhibited by heterocyclic sulfonamides in a manner similar to native CA I, its affinity for several aromatic sulfonamides was lower [35]. We have been recently able to determine the crystal structure of CA I M1 and to identify the presence of a second zinc binding site in its active cavity which is seemingly involved in the previously reported metal ion activation of naphthyl acetates hydrolysis [36]. These observations prompted us to investigate the differences in the interaction of aromatic sulfonamides with the CA I M1 variant and its (Zn)2 adduct. The crystal structures presented in this paper give us new insights into the inhibition mechanism of this
carbonic anhydrase isoenzyme, and on the effect of the second Zn(II) ion on sulfonamide binding.
2. Experimental
2.1. Materials Buffers, 4-nitrophenylacetate, b-naphthyl acetate, acetonitrile, were from Sigma-Aldrich and used without further purification. All the other chemicals were of the best purity available. All buffers used in the kinetic measurements were brought to an ionic strength m/0.1, by addition of Na2SO4. The cDNA encoding human CA I M1 was produced from a human placental cDNA library by site-directed mutagenesis employing the overlap extension method [37]. This was cloned into plasmid pKK233-2 which was then transformed into Escherichia coli JM109 cells. These were grown in LB medium containing 50 mg ml 1 of ampicillin and enzyme synthesis was induced by the addition of 0.5 mM isopropyl thio-b-D-galactoside and 0.5 mM ZnSO4, followed by incubation at 30 8C for 8/12 h. CA I M1 was purified by affinity chromatography, using p -aminomethylbenzene sulfonamide (PAMBS) [38]. Enzyme concentrations were determined spectrophotometrically using o 280 /49 mM 1 cm 1 based on MW/30 000 Da. Electrophoresis on a 10% polyacrylamide gel stained with Coomassie blue was used to confirm purity, which was greater than 95%.
2.2. Enzymatic assay The catalytic activity of the purified enzyme was checked by measuring the initial rates of hydrolysis of 4nitrophenyl acetate at 25 8C. The assay medium contained 0.4 mM substrate, 10% (v/v) acetonitrile and 50 mM Tris /H2SO4 and 50 mM Na2SO4 buffer, pH 8.0 [28]. The formation of product was monitored at the isosbestic point for the corresponding nitrophenol and nitrophenolate ion (348 nm). The apparent second-order rate constants, kenz (kcat/Km), were calculated using o 348 /5.15 mM 1 cm 1 for the reaction with 4-nitrophenyl acetate. Nonenzymic rates, estimated from blank reactions with all components except enzyme, were subtracted from the observed, total initial rates. Solutions of substrate were prepared in anhydrous acetonitrile; the substrate concentrations varied between 2/ 102 and 1/106 M. CA inhibition was measured spectrophotometrically with 4-nitrophenyl acetate as substrate, at 25 8C and pH 8 [39].
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2.3. Protein crystallization and data collection Crystals of CA I M1 were obtained using the hanging drop vapor diffusion method. Five microliters of a 14 mg ml1 protein solution in 10 mM Tris /SO4 were mixed with 5 ml of a precipitant solution containing 25% PEG 4000, LiCl 0.4 M, Tris /HCl 100 mM pH 9.0, 10% ethylene glycol. In order to obtain the CA I M1 complex with sulfanilamide, crystals of the enzyme were soaked in a solution containing 25% PEG 4000, 0.4 M LiCl, Hepes 100 mM pH 9.09, ethylene glycol 10%, sulfanilamide 5 mM for 1 day. Crystals of the CA I M1 (Zn)2 adduct with sulfanilamide were obtained by soaking the crystals of the enzyme in a solution containing 25% PEG 4000, LiCl 0.4 M, Tris /HCl 100 mM pH 9.0, 10% ethylene glycol, ZnSO4 5 mM for 5 h and then in another solution containing sulfanilamide 5 mM as previously described for 1 day. Data were collected at 100 K on both complexes using a Cu Ka radiation from a rotating anode X-ray generator coupled with a SMART 1K CCD detector from Bruker and were processed using SAINT. The crystals were isomorphous with the CA I M1 [36]. Further details of the data collection and processing are reported in Table 1. The stereochemically restrained refinement was carried out with the program Refmac from the CCP4 program suite and the Rfree was used to follow the progress of the refinement [40]. The CA 1 M1 structure (Protein Data Bank entry 1j9w [41]), without solvent molecules was used as an initial model. The 2Fo-Fc and Fo-Fc difference maps showed density close to the catalytic zinc in both the Table 1 Data collection and processing statistics Sulfanilamide /CA I M1 adduct Crystal system Space group Molecules/asymmetric unit Unit cell dimension ˚) a (A ˚) b (A ˚) c (A Resolution range ˚) (A Raw measurements Unique reflections Completeness Rsymm (%) Average I /s (I ) Redundancy
Sulfanilamide /CA I M1 (Zn)2 adduct
orthorombic P 212121 2
61.73 71.15 120.37 20 /2.2
62.42 71.39 121.25 20 /2.6
43378 25210 90.2 9.7 8.6 1.7
55656 17003 100.0 8.8 6.2 3.3
137
molecules in the asymmetric units and in both complexes that could be attributed to a sulfamido group. When this group was fitted in the density and the model was subjected to cycles of refinement then the difference maps showed density also for the aromatic ring of the inhibitor. A model for the sulfanilamide molecule was fitted in the 2Fo-Fc maps contoured at 1s level. The coordinates were subjected to rounds of refinement calculations interspersed with sessions of model fitting using Quanta on a O2 Silicon Graphics workstation [42]. Solvent molecules were introduced in the model using ARP [43]. Anisotropy was introduced in the refinement of the temperature factors for the metal ions and sulfur atoms for both the complex structures. The main chain atoms in the two molecules in the asymmetric unit were subjected to non crystallographic symmetry restraints. The Statistics for refinements are reported in Table 2.
3. Results and discussion CA I M1 was the first variant of a CA isozyme to be described. It is of particular interest for its special catalytic properties such as the zinc ion activated hydrolysis of a- and b-naphthyl acetates [32,44]. The three-dimensional structure of the human CA I M1 and of its (Zn)2 adduct has recently been determined [36]. The global structure was not altered by the single point mutation His67Arg, but the mutated residue shows a distinct orientation, pointing towards the external part of the active site contrarily to His67 in the wild type enzyme which is oriented towards the interior part of the cavity [3,36]. Also the hydrogen bond network involved in the catalytic process appear to be altered by this single point mutation although this does not seem to affect the catalytic rates for the CO2 hydration reaction [36]. Furthermore, it has also been observed that this mutation is able to trigger the binding of a second zinc(II) ion into the active site which seems to be responsible for the tenfold activation of the hydrolysis of a- and b-naphthyl acetates. In the CAI M1/(Zn)2 adduct the side chain of the mutated residue Arg67 moves towards the interior part of the cavity, entering the coordination sphere of the second zinc ion, together with the side chains of His 200 and His 64. Other studies from our laboratory have also revealed that, although CA I M1 was inhibited by heterocyclic sulfonamides to a similar extent to wild type CA I, its affinity for several aromatic sulfonamides was much lower [35]. These observations prompted us to further analyze the effect of such mutation on sulfonamide inhibitors binding.
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138 Table 2 Structure refinement statistics
Resolution limits Proteins atoms Water atoms Metal ions and others R (%) Rfree (%) Stereochemistry ˚) Bond length (A ˚) Angle distance (A ˚) Planar 1 /4 distance (A ˚ 2) Average B-factor (A
Weight 0.020 0.040 0.050 Total Mol A Mol B Solvents
Sulfanilamide /CA I M1 adduct
Sulfanilamide /CA I M1 (Zn)2 adduct
20.0 /2.2 3997 465 28 18.9 26.8 Rms 0.008 0.039 0.077 21.25 21.37 21.13 30.85
20.0 /2.6 4002 280 26 21.4 28.5
Inhibition data with aromatic/heterocyclic sulfonamides against CA I M1, human CA I, human CA II and bovine CA IV are shown in Table 3. Wild type CA I has a decreased affinity for sulfonamide inhibitors as compared with the fast isozyme CA II. The variant form, CA I M1 possesses even a lower affinity for several aromatic sulfonamides, whereas it is appreciably inhibited by heterocyclic sulfonamides, in a manner similar to wild type CA I. Thus, acetazolamide, benzolamide and ethoxzolamide possess high affinities both for wild type as well as CA I M1 (KI-s in the range 108 /109 M, with CA I M1 only slightly less inhibited than wild type CA I). Aromatic sulfonamides such as sulfanilamide, orthanilamide, p-aminomethyl- and p-aminoethyl-benzenesulfonamide on the other hand had a 10 /20 times lower affinity for CA I M1 as compared with wild type CA I. A similar difference in affinity for sulfonamide inhibitors has been recently reported between the physiologically important isozymes CA II and CA IV (5), with CA
0.007 0.038 0.063 22.84 22.60 22.22 29.07
IV possessing 15/30 times lower affinity as compared with CA II for this type of inhibitors. This behavior is critical in medical treatments based on sulfonamide CA inhibitors (such as the topical antigaucoma therapy) since the target isozymes to be inhibited are CA II, IV and probably also CA I [12 /16]. Here we report the crystal structures of the sulfanilamide */(structure 8 in Scheme 1) CA I M1 and sulfanilamide */CA I M1 (Zn)2 adducts. The present crystal structures are compared with the structure of native human CA I complexed with amsulf (3acetoxymercuri-4-aminobenzene sulfonamide) [22,23]. Since the 3-acetoxymercuri-fragment of amsulf is released upon binding to the enzyme, the resulting inhibitor molecule is identical to sulfanilamide. The crystal structure of CA I M1 revealed the occurrence of two independent molecules in the asymmetric unit [36]. The changes observed upon inhibitor binding to both molecules as well as the differences between molecules A and B will be described in detail.
Table 3 Inhibition data with sulfonamide inhibitors 1 /11 against isozymes CA I M1; CA I, CA II, and CA IV Number Inhibitor
1 2 3 4 5 6 7 8 9 10 11 a b c
Acetazolamide Benzolamide Ethoxzolamide Dorzolamide Dichlorophenamide Orthanilamide Metanilamide Sulfanilamide 4-Hydrazino-benzenesulfonamide p -aminomethyl-benzenesulfonamide p -Aminoethyl-benzenesulfonamide
KI
a
CA I M1 (mM) [present work]
CA I
1.30 0.080 0.090 55 2.10 330 510 540 830 260 245
0.90 0.015 0.025 50.0 1.20 45.4 25.0 28.0 78.5 25.0 21.0
b
(mM) [45]
Standard error for the determination of KI was 5 /10% (average from three different assays). Human (cloned) isozyme. Isolated from bovine lung microsomes.
CA II 12 9 8 9 38 295 240 300 320 170 160
a
(nM) [45]
CA IV 220 12 13 45 380 1310 2200 3000 3215 2800 2450
c
(nM) [45]
M. Ferraroni et al. / Inorganica Chimica Acta 339 (2002) 135 /144
139
Scheme 1. Structure of sulfonamide inhibitors 1-11 investigated in the present study.
Fig. 1. Least-squares superimposition of the most relevant active site residues of the wild-type Human CA I (His67) (dark structure) and the CA I M1 (Arg67) (light structure) molecule B, involved in sulfonamide inhibitor binding. Bound sulfanilamide is also shown.
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Fig. 1 shows the least-squares superimposition of the most relevant active site residues of the wild-type CA I (His67) (dark/blue structure) and the CA I M1 (Arg67) molecule B (bright/yellow structure) involved in sulfonamide inhibitor binding. In all cases the N1 nitrogen of sulfanilamide has displaced the zinc bound water/ hydroxide moiety and its O1 and O2 oxygens have taken the place of other solvent molecules present in the active site. The bond distances and angles of the catalytic zinc ion coordination polyhedron in the sulfanilamide /CA I M1 adduct are reported in Table 4. The noteworthy distances among sulfanilamide atoms and the atoms of the neighbouring residues are reported in Table 5. Significant changes are observed in the sulfanilamide molecule positioning, particularly regarding the p -amino group which is shifted away from residue Leu198, towards OE1 of Gln92 and consequently Arg67. This is possibly a result of the different conformation of the Gln92 side chain in CA I M1 with respect to the wild type isozyme (see Fig. 1). Comparing the wild type CA I amsulf adduct with molecule B of the CA I M1 /sulfanilamide adduct it appears that the sulfamido N1 /Zn(II) bond distance is ˚ . As a consequence, the van changed from 1.86 to 1.93A der Waals interaction between Thr199 OG1 and sulfa˚ ; and the mido O2 is shortened from 3.85 to 3.73 A hydrogen bond between Thr199 OG1 and sulfamido N1 ˚. is reduced from 3.25 to 2.84 A Therefore, in molecule B the p -amino moiety of the inhibitor is bent towards Arg67 due to the stronger interaction with Gln92 thus resulting in a longer bond distance between the metal ion and the N1 atom of the sulfamido group of sulfanilamide, partly counterbalanced by shorter hydrogen bonds with Thr199. The picture appears to be significantly different for molecule A (see Fig. 2), where the hydrogen bond interaction of sulfanilamide N2 with an exogenous ethylene glycol molecule, present in the crystallization buffer, seems to favour a stronger binding of the sulfamido group to the catalytic zinc ion and to Thr199 OG1. Indeed, in this case, the sulfamido N1 / ˚ (1.86 A ˚ in the wild type Zn(II) bond distance is 1.84 A CA I adduct). The van der Waals interaction between ˚ ) as well as the Thr199 OG1 and sulfamido O2 (3.40 A hydrogen bond between Thr199 OG1 and sulfamido N1 ˚ ) are also shorter than in molecule B (see Table (2.80 A 5). Since the inhibition constants were measured in the absence of ethylene glycol, we consider that the picture observed in molecule B is more consistent with such data. The overall differences observed between Molecule B and the wild type CA I, in particular the weaker bond of the inhibitor with the active metal ion could explain the reduced inhibition constants with aromatic sulfona-
mides observed for CA I M1 with respect to wild type CA I. We also examined the effect of the formation of the (Zn)2 adduct on sulfanilamide binding to CA I M1. The least-squares superimposition of the most relevant active site residues involved in sulfanilamide inhibitor binding to the CA I M1 (light/yellow structure) and its (Zn)2 adduct (dark/red structure) is reported in Fig. 3.
Fig. 2. Refined models of the sulfanilamide molecule and the interacting active site region for molecules A and B of CA1 M1 adducts.
˚) Distance X Zn (A CA I M1
His 94 No2 His 96 No2 His 119 Nd2 N1 (Sulfanilamide)
X Zn H94 CA I
Mol A
Mol B
1.93 2.01 2.01 1.84
1.91 2.03 2.01 1.93
CA I M1 Mol A
2.05 2.14 1.97 1.86
X Zn H96 CA I
Mol B
CA I M1
X Zn H119 CA I
Mol A
Mol B
102.1
101.9
105.4
CA I M1
X Zn N1 (sulfanilamide) CA I
Mol A
Mol B
116.3 95.3
114.3 96.5
113.3 96.9
CA I M1
CA I
Mol A
Mol B
114.0 111.6 114.9
117.6 110.4 113.2
108.1 118.4 114.3
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Table 4 Bond distances and angles of the catalytic Zn ion coordination polyhedron in the Sulfanilamide /CA I M1 adduct
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Fig. 3. Least-squares superimposition of the most relevant active site residues of the CA I M1 (light structure) and the CA I M1 (Zn)2 adduct (dark structure) molecule B, involved in sulfonamide inhibitor binding. Bound sulfanilamide is also shown. Table 5 Neighbors of Sulfanilamide Molecule in CA I, CA I M1, and CA I M1× Zn(II)2 adducts Sulfanilamide atom
N1 O1 O2 N2
Neighbor/atom
Zn(II) Thr199 OG1 Zn Thr199 OG1 Gln92 OE1 Gln92 NE2 Arg67 NH1 Arg67 NH2 EGL O2
Distances in CA I wild type
1.86 3.25 3.00 3.85 5.27 5.04
Distances in CA I M1
Distances in CA I M1 × Zn(II)2
Mol A
Mol B
Mol A
Mol B
1.84 2.80 3.07 3.40 4.59 6.47 7.42 5.43 3.11
1.93 2.84 3.07 3.73 4.23 6.37 7.37 5.45
1.74 2.97 3.35 3.51 3.38 5.32 4.45 4.23
1.93 2.65 2.97 3.64 3.42 5.62 3.43 3.10
Table 6 Bond Distances and Angles of both Zn Ion Coordination Polyhedrons in the Sulfanilamide-CA I M1× Zn(II)2 adduct
His 200 No2 His 64 No2 Arg 67 Wat241 O
His 94 No2 His 96 No2 His 119 Nd2 Sulfanilamide N1
˚) Distance X Zn (A
X Zn H200
X Zn H64
X Zn R67
X Zn Wat241 O
Mol A
Mol B
Mol A
Mol A
Mol B
Mol A
Mol B
Mol A
Mol B
1.92 2.25 1.91 2.23
2.18 2.20 2.08 2.11
104.6
104.2
114.8 116.0
118.6 105.9
97.4 114.1 108.5
106.1 112.9 109.3
1.87 2.10 2.03 1.74
2.01 1.97 2.00 1.93
Mol B
X Zn H96
X Zn 119
X Zn Sulfanilamide N1
Mol A
Mol B
Mol A
Mol B
Mol A
Mol B
94.2
97.4
113.4 100.0
109.0 103.1
118.3 117.9 110.9
115.0 119.1 111.6
M. Ferraroni et al. / Inorganica Chimica Acta 339 (2002) 135 /144
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˚ ). The hydrogen bond between Thr199 OG1 and (3.42 A ˚ and with sulfanilamide N1 passes from 2.84 to 2.65 A ˚ . The sulfanilamide sulfanilamide O2 from 3.73 to 3.64 A N1 /Zn(II) bond distance is not further changed upon ˚ ). formation of the (Zn)2 adduct (1.93 A The tilting of the inhibitor molecule towards Gln92 and Arg67 is accompanied by small movements of Leu198 and His200 which appear to be further pushed in the opposite directions. The main differences observed in molecule A are the further shortening of the sulfanilamide N1 /Zn(II) bond ˚ ) counterbalanced by a longer Thr199 distance (1.74 A ˚ ) interaction. On the OG1 /sulfanilamide N1 (2.97 A opposite side of the inhibitor molecule the much longer distances between sulfanilamide N2 and Arg67 NH2 ˚ ) and NH1 (4.23 A ˚ ) are worthy of note. This is (4.45 A probably associated to the presence of a water molecule (Wat 179 in Fig. 4(A)) which has a strong van der Waals ˚ ). interaction with Arg67 (3.39 A Fluorescence experiments on the dansylamide (5(dimethylamino)naphthalene-1-sulfonamide) /CA I M1 adduct suggested that the presence of additional zinc ions induced the inhibitor molecule to move towards a more hydrophobic environment [32]. The present data show that, at least in the case of sulfanilamide, the presence of the second zinc induces a further shift of the inhibitor molecule towards the more hydrophilic site of the active site cavity, thus moving away from the hydrophobic region of Leu198. Dansylamide is certainly bulkier than sulfanilamide and therefore it is, at the moment, unfeasible to ascertain the possible orientational changes for such an inhibitor. Further experiments to investigate the inhibition of the CA I M1 (Zn)2 adduct by bulkier sulfonamides are currently in progress.
Acknowledgements Fig. 4. Refined models of the sulfanilamide molecule and the interacting active site region for molecules A and B of CA1 M1 (Zn)2 adducts.
The bond distances and angles of both zinc ion coordination polyhedrons in the sulfanilamide /CA I M1 (Zn)2 adduct are reported in Table 6. Upon binding of the second Zn(II) ion, the Arg67 side-chain moves towards the cavity, entering the second metal ion coordination sphere, and its rearrangement triggers a further repositioning of the inhibitor molecule. In molecule B, the p -amino moiety of the inhibitor establishes a new hydrogen bond with Arg67 NH2 (3.10 ˚ ) and a van der Waals interaction with Gln92 OE1 A
The authors of the present article want to acknowledge the valuable scientific contributions in bioinorganic chemistry of Helmut Sigel during his career, but also to thank him and his wife, Astrid, for the impressive editorial and organizational activity in favour of the bioinorganic community. We feel that a large part of our formation has been created along the years by the availability of fundamental book series and handbooks edited by them. The roles of Dr. R.E. Tashian and Dr. K.E. Wiebauer in the original discovery of the HCA I M1 variant and in the subsequent production of the cDNA clone, respectively, are gratefully acknowledged. We also wish to thank the Target Project on Biotechnologies CNR Italy.
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[25] P.A. Boriack-Sjodin, S. Zeitlin, H.H. Chen, L. Crenshaw, S. Gross, A. Dantanarayana, P. Delgado, J.A. May, T. Dean, D.W. Christianson, Protein Sci. 7 (1998) 2483. [26] F. Briganti, V. Iaconi, S. Mangani, P. Orioli, A. Scozzafava, G. Vernaglione, C.T. Supuran, Inorg. Chim. Acta 275 /276 (1998) 295. [27] F. Briganti, S. Mangani, P. Orioli, A. Scozzafava, G. Vernaglione, C.T. Supuran, Biochem. 36 (1997) 10384. [28] I.D. Campbell, S. Lindskog, A.I. White, J. Mol. Biol 90 (1974) 469. [29] C. Engstrand, B.H. Jonsson, S. Lindskog, Eur. J. Biochem. 229 (1995) 696. [30] P.B. Venta, in: W.R. Chegwidden, N.D. Carter, Y.H. Edwards (Eds.), The Carbonic Anhydrases: New Horizons, Birkhauser, Switzerland, 2000, p. 403. [31] C.R. Shaw, F.N. Syner, R.E. Tashian, Science 138 (1962) 31. [32] R.E. Tashian, N.D. Carter, Adv. Hum. Genet. 7 (1976) 1. [33] R.E. Tashian, A.G. Kendall, N.D. Carter, Hemoglobin 4 (1980) 6350. [34] W.R. Chegwidden, L.E. Wagner, P.J. Venta, N.C.H. Bergenhem, Y.S.L. Yu, R.E. Tashian, Human Mutat. 4 (1994) 294. [35] F. Briganti, W.R. Chegwidden, A. Scozzafava, C.T. Supuran, R.E. Tashian, K.E. Wiebauer, Gene Fam. Isozyme Bull. 31 (1998) 43. [36] M. Ferraroni, F. Briganti, W.R. Chegwidden, C.T. Supuran, K.E. Wiebauer, R.E. Tashian, A. Scozzafava, Biochem. 41 (2002) 6237. [37] W.R. Chegwidden, K.E. Wiebauer, P.J. Venta, N.C.H. Bergenhem, R.E. Tashian, Isozyme Bull. 28 (1995) 37. [38] R.G. Khalifah, D.J. Strader, S.H. Bryant, S.M. Gibson, Biochem. 16 (1977) 2241. [39] Y. Pocker, J.T. Stone, Biochem. 6 (1967) 668. [40] Collaborative Computational Project, N. 4, Acta Crystallogr., D 50 (1994) 760 (Abstract). [41] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res. 28 (2000) 235. [42] Quanta Simulation, Search, and Analysis, Molecular Simulations Inc., San Diego, July 1997. [43] V.S. Lamzin, K.S. Wilson, Acta Crystallogr., D 49 (1993) 129. [44] R.E. Tashian, The Carbonic Anhydrases */New Horizons, Birkha¨user Verlag, Basel, 2000, p. 569. [45] A. Scozzafava, L. Menabuoni, F. Mincione, F. Briganti, G. Mincione, C.T. Supuran, J. Med. Chem. 43 (2000) 4542.
Crystal analysis of aromatic sulfonamide binding to native and (Zn)2 adduct of human carbonic anhydrase I Michigan 1 Marta Ferraroni a, Fabrizio Briganti a, W. Richard Chegwidden b, Claudiu T. Supuran a, Andrea Scozzafava a,* a
Laboratorio di Chimica Bioinorganica, Dipartimento di Chimica, Universita` degli Studi di Firenze, Via della Lastruccia, 3, I-50019 Sesto Fiorentino, Italy b Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16509, USA Received 4 November 2001; accepted 30 January 2002 Dedicated in honor of Professor Helmut Sigel
Abstract The crystal structures of sulfanilamide (4-aminobenzene-sulfonamide) complexed to the enzyme variant human carbonic ˚ resolution respectively are described. anhydrase I Michigan 1 (CA I M1) and to its (Zn)2 adduct refined at 2.2 and 2.6 A Comparisons among these structures and the corresponding sulfonamide adduct of human CA I show significant differences in the orientation of the inhibitor molecule and in its interactions with active site residues such as His200, Thr199, Leu198, Gln92, and Arg/His67 which are known to play important roles in substrate or inhibitor recognition and binding. In CA I M1 molecule B a lengthening of the Zn/N1 sulfanilamide bond distance and a corresponding shortening of the distance between the sulfonamido group and Thr199 are observed compared with the values for native CA I. When the second Zn(II) ion is present in the active site, the p -amino group and the aromatic ring of the inhibitor molecule appear to tilt towards Gln92 and Arg67, moving away from residues His200 and Leu198. The structural differences in inhibitor binding between the CA I isozyme and the CA I M1 variant are discussed in terms of the different inhibition constants measured for a variety of aromatic and heterocyclic sulfonamides. # 2002 Elsevier Science B.V. All rights reserved. Keywords: X-ray crystallography; Zinc enzymes; Carbonic anhydrase; Genetic variant; Point mutation CA I Michigan 1; Sulfonamides; Sulfanilamide; Inhibition
1. Introduction Carbonic anhydrase isoenzymes (CA, EC 4.2.1.1) are seemingly ubiquitous, well characterized metalloenzymes that are known to occur in three gene families a, b and g. The human isozymes all belong to the afamily and are monomeric, containing one zinc ion per polypeptide chain (Mr /30 kDa) [1]. These highly abundant proteins are involved in critical physiological processes connected with respiration and transport of CO2/HCO3 between metabolizing tissues and the lungs, pH homeostasis, electrolyte Abbreviations: CA, Carbonic anhydrase; HCA, Human carbonic anhydrase; CA I M1, Carbonic anhydrase I Michigan 1. * Corresponding author. Fax: /39-055-457 3385 E-mail address: [email protected] (A. Scozzafava).
secretion in a variety of tissues/organs, and biosynthetic reactions, such as gluconeogenesis, lipogenesis and ureagenesis [1,2]. The X-ray structures of some isozymes have shown that the zinc ion is coordinated to three histidines and a water molecule/hydroxide ion [3 /7]. The catalytic mechanism of CAs has been extensively investigated through kinetic, spectroscopic and structural studies [8 /10]. The enzyme can also act on other carbonyl systems, such as esters and aldehydes [11]. Catalysis of both ester hydrolysis and aldehyde hydration appears to occur by the zinc-hydroxide mechanism as for the CO2 hydration reaction [10]. Inhibition of several of these enzymes by aromatic/ heterocyclic sulfonamides has been exploited clinically for more than 45 years in the treatment of a variety of
0020-1693/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 0 - 1 6 9 3 ( 0 2 ) 0 0 9 5 9 - 3
136
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diseases such as glaucoma, epilepsy, congestive heart failure, mountain sickness, gastric and duodenal ulcers [12 /16]. The inhibitory mechanism is primarily controlled by the coordination of the sulfonamide group to the catalytic zinc ion and secondarily by finely tuned tight binding to other regions of the active site cavity [17 /25]. A variety of CA activators has also been recently investigated and the structural characterization of some of their adducts with CA II has led to the identification of a novel ‘activator binding site’ in the CA cavity [26,27]. Many mutations of active-site residues of human carbonic anhydrase (HCA) II resulting in changes of CO2 hydration as well as of esterase activity and inhibitor-binding properties have been extensively studied, permitting identification of the amino acid residues that play important roles in catalysis. In CA I, which possesses about one fifth of the activity of CA II, the situation is less clear; the pKa of His 64 is too low for efficient proton transfer and the specific pathway or pathways of protons involving shuttle groups remain to be established [10,28]. Several active-site mutants of CA I have been prepared by site-directed mutagenesis in order to try to understand the striking catalytic differences between CA I and CA II, the two isozymes that predominate in the red cell [29]. A number of naturally occurring variants of mammalian CA isozymes have also been reported [30]. One variant of human CA I (CA I Michigan 1) has attracted particular interest, since it provides an example of a point mutation that results in true metal ion activation of the gene product [31]. Although its CO2 hydration and esterase activities appeared to be within the normal range, its esterase activity toward a- or b-naphthyl acetate was enhanced tenfold at a Zn(II) concentration of 10 4 M [32,33]. The elusive mutation conferring the above reported properties was recently revealed by Chegwidden et al. and turned out to be His67Arg [34]. Other studies from our laboratory have also revealed that, although CA I Michigan 1 (CA I M1) was inhibited by heterocyclic sulfonamides in a manner similar to native CA I, its affinity for several aromatic sulfonamides was lower [35]. We have been recently able to determine the crystal structure of CA I M1 and to identify the presence of a second zinc binding site in its active cavity which is seemingly involved in the previously reported metal ion activation of naphthyl acetates hydrolysis [36]. These observations prompted us to investigate the differences in the interaction of aromatic sulfonamides with the CA I M1 variant and its (Zn)2 adduct. The crystal structures presented in this paper give us new insights into the inhibition mechanism of this
carbonic anhydrase isoenzyme, and on the effect of the second Zn(II) ion on sulfonamide binding.
2. Experimental
2.1. Materials Buffers, 4-nitrophenylacetate, b-naphthyl acetate, acetonitrile, were from Sigma-Aldrich and used without further purification. All the other chemicals were of the best purity available. All buffers used in the kinetic measurements were brought to an ionic strength m/0.1, by addition of Na2SO4. The cDNA encoding human CA I M1 was produced from a human placental cDNA library by site-directed mutagenesis employing the overlap extension method [37]. This was cloned into plasmid pKK233-2 which was then transformed into Escherichia coli JM109 cells. These were grown in LB medium containing 50 mg ml 1 of ampicillin and enzyme synthesis was induced by the addition of 0.5 mM isopropyl thio-b-D-galactoside and 0.5 mM ZnSO4, followed by incubation at 30 8C for 8/12 h. CA I M1 was purified by affinity chromatography, using p -aminomethylbenzene sulfonamide (PAMBS) [38]. Enzyme concentrations were determined spectrophotometrically using o 280 /49 mM 1 cm 1 based on MW/30 000 Da. Electrophoresis on a 10% polyacrylamide gel stained with Coomassie blue was used to confirm purity, which was greater than 95%.
2.2. Enzymatic assay The catalytic activity of the purified enzyme was checked by measuring the initial rates of hydrolysis of 4nitrophenyl acetate at 25 8C. The assay medium contained 0.4 mM substrate, 10% (v/v) acetonitrile and 50 mM Tris /H2SO4 and 50 mM Na2SO4 buffer, pH 8.0 [28]. The formation of product was monitored at the isosbestic point for the corresponding nitrophenol and nitrophenolate ion (348 nm). The apparent second-order rate constants, kenz (kcat/Km), were calculated using o 348 /5.15 mM 1 cm 1 for the reaction with 4-nitrophenyl acetate. Nonenzymic rates, estimated from blank reactions with all components except enzyme, were subtracted from the observed, total initial rates. Solutions of substrate were prepared in anhydrous acetonitrile; the substrate concentrations varied between 2/ 102 and 1/106 M. CA inhibition was measured spectrophotometrically with 4-nitrophenyl acetate as substrate, at 25 8C and pH 8 [39].
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2.3. Protein crystallization and data collection Crystals of CA I M1 were obtained using the hanging drop vapor diffusion method. Five microliters of a 14 mg ml1 protein solution in 10 mM Tris /SO4 were mixed with 5 ml of a precipitant solution containing 25% PEG 4000, LiCl 0.4 M, Tris /HCl 100 mM pH 9.0, 10% ethylene glycol. In order to obtain the CA I M1 complex with sulfanilamide, crystals of the enzyme were soaked in a solution containing 25% PEG 4000, 0.4 M LiCl, Hepes 100 mM pH 9.09, ethylene glycol 10%, sulfanilamide 5 mM for 1 day. Crystals of the CA I M1 (Zn)2 adduct with sulfanilamide were obtained by soaking the crystals of the enzyme in a solution containing 25% PEG 4000, LiCl 0.4 M, Tris /HCl 100 mM pH 9.0, 10% ethylene glycol, ZnSO4 5 mM for 5 h and then in another solution containing sulfanilamide 5 mM as previously described for 1 day. Data were collected at 100 K on both complexes using a Cu Ka radiation from a rotating anode X-ray generator coupled with a SMART 1K CCD detector from Bruker and were processed using SAINT. The crystals were isomorphous with the CA I M1 [36]. Further details of the data collection and processing are reported in Table 1. The stereochemically restrained refinement was carried out with the program Refmac from the CCP4 program suite and the Rfree was used to follow the progress of the refinement [40]. The CA 1 M1 structure (Protein Data Bank entry 1j9w [41]), without solvent molecules was used as an initial model. The 2Fo-Fc and Fo-Fc difference maps showed density close to the catalytic zinc in both the Table 1 Data collection and processing statistics Sulfanilamide /CA I M1 adduct Crystal system Space group Molecules/asymmetric unit Unit cell dimension ˚) a (A ˚) b (A ˚) c (A Resolution range ˚) (A Raw measurements Unique reflections Completeness Rsymm (%) Average I /s (I ) Redundancy
Sulfanilamide /CA I M1 (Zn)2 adduct
orthorombic P 212121 2
61.73 71.15 120.37 20 /2.2
62.42 71.39 121.25 20 /2.6
43378 25210 90.2 9.7 8.6 1.7
55656 17003 100.0 8.8 6.2 3.3
137
molecules in the asymmetric units and in both complexes that could be attributed to a sulfamido group. When this group was fitted in the density and the model was subjected to cycles of refinement then the difference maps showed density also for the aromatic ring of the inhibitor. A model for the sulfanilamide molecule was fitted in the 2Fo-Fc maps contoured at 1s level. The coordinates were subjected to rounds of refinement calculations interspersed with sessions of model fitting using Quanta on a O2 Silicon Graphics workstation [42]. Solvent molecules were introduced in the model using ARP [43]. Anisotropy was introduced in the refinement of the temperature factors for the metal ions and sulfur atoms for both the complex structures. The main chain atoms in the two molecules in the asymmetric unit were subjected to non crystallographic symmetry restraints. The Statistics for refinements are reported in Table 2.
3. Results and discussion CA I M1 was the first variant of a CA isozyme to be described. It is of particular interest for its special catalytic properties such as the zinc ion activated hydrolysis of a- and b-naphthyl acetates [32,44]. The three-dimensional structure of the human CA I M1 and of its (Zn)2 adduct has recently been determined [36]. The global structure was not altered by the single point mutation His67Arg, but the mutated residue shows a distinct orientation, pointing towards the external part of the active site contrarily to His67 in the wild type enzyme which is oriented towards the interior part of the cavity [3,36]. Also the hydrogen bond network involved in the catalytic process appear to be altered by this single point mutation although this does not seem to affect the catalytic rates for the CO2 hydration reaction [36]. Furthermore, it has also been observed that this mutation is able to trigger the binding of a second zinc(II) ion into the active site which seems to be responsible for the tenfold activation of the hydrolysis of a- and b-naphthyl acetates. In the CAI M1/(Zn)2 adduct the side chain of the mutated residue Arg67 moves towards the interior part of the cavity, entering the coordination sphere of the second zinc ion, together with the side chains of His 200 and His 64. Other studies from our laboratory have also revealed that, although CA I M1 was inhibited by heterocyclic sulfonamides to a similar extent to wild type CA I, its affinity for several aromatic sulfonamides was much lower [35]. These observations prompted us to further analyze the effect of such mutation on sulfonamide inhibitors binding.
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138 Table 2 Structure refinement statistics
Resolution limits Proteins atoms Water atoms Metal ions and others R (%) Rfree (%) Stereochemistry ˚) Bond length (A ˚) Angle distance (A ˚) Planar 1 /4 distance (A ˚ 2) Average B-factor (A
Weight 0.020 0.040 0.050 Total Mol A Mol B Solvents
Sulfanilamide /CA I M1 adduct
Sulfanilamide /CA I M1 (Zn)2 adduct
20.0 /2.2 3997 465 28 18.9 26.8 Rms 0.008 0.039 0.077 21.25 21.37 21.13 30.85
20.0 /2.6 4002 280 26 21.4 28.5
Inhibition data with aromatic/heterocyclic sulfonamides against CA I M1, human CA I, human CA II and bovine CA IV are shown in Table 3. Wild type CA I has a decreased affinity for sulfonamide inhibitors as compared with the fast isozyme CA II. The variant form, CA I M1 possesses even a lower affinity for several aromatic sulfonamides, whereas it is appreciably inhibited by heterocyclic sulfonamides, in a manner similar to wild type CA I. Thus, acetazolamide, benzolamide and ethoxzolamide possess high affinities both for wild type as well as CA I M1 (KI-s in the range 108 /109 M, with CA I M1 only slightly less inhibited than wild type CA I). Aromatic sulfonamides such as sulfanilamide, orthanilamide, p-aminomethyl- and p-aminoethyl-benzenesulfonamide on the other hand had a 10 /20 times lower affinity for CA I M1 as compared with wild type CA I. A similar difference in affinity for sulfonamide inhibitors has been recently reported between the physiologically important isozymes CA II and CA IV (5), with CA
0.007 0.038 0.063 22.84 22.60 22.22 29.07
IV possessing 15/30 times lower affinity as compared with CA II for this type of inhibitors. This behavior is critical in medical treatments based on sulfonamide CA inhibitors (such as the topical antigaucoma therapy) since the target isozymes to be inhibited are CA II, IV and probably also CA I [12 /16]. Here we report the crystal structures of the sulfanilamide */(structure 8 in Scheme 1) CA I M1 and sulfanilamide */CA I M1 (Zn)2 adducts. The present crystal structures are compared with the structure of native human CA I complexed with amsulf (3acetoxymercuri-4-aminobenzene sulfonamide) [22,23]. Since the 3-acetoxymercuri-fragment of amsulf is released upon binding to the enzyme, the resulting inhibitor molecule is identical to sulfanilamide. The crystal structure of CA I M1 revealed the occurrence of two independent molecules in the asymmetric unit [36]. The changes observed upon inhibitor binding to both molecules as well as the differences between molecules A and B will be described in detail.
Table 3 Inhibition data with sulfonamide inhibitors 1 /11 against isozymes CA I M1; CA I, CA II, and CA IV Number Inhibitor
1 2 3 4 5 6 7 8 9 10 11 a b c
Acetazolamide Benzolamide Ethoxzolamide Dorzolamide Dichlorophenamide Orthanilamide Metanilamide Sulfanilamide 4-Hydrazino-benzenesulfonamide p -aminomethyl-benzenesulfonamide p -Aminoethyl-benzenesulfonamide
KI
a
CA I M1 (mM) [present work]
CA I
1.30 0.080 0.090 55 2.10 330 510 540 830 260 245
0.90 0.015 0.025 50.0 1.20 45.4 25.0 28.0 78.5 25.0 21.0
b
(mM) [45]
Standard error for the determination of KI was 5 /10% (average from three different assays). Human (cloned) isozyme. Isolated from bovine lung microsomes.
CA II 12 9 8 9 38 295 240 300 320 170 160
a
(nM) [45]
CA IV 220 12 13 45 380 1310 2200 3000 3215 2800 2450
c
(nM) [45]
M. Ferraroni et al. / Inorganica Chimica Acta 339 (2002) 135 /144
139
Scheme 1. Structure of sulfonamide inhibitors 1-11 investigated in the present study.
Fig. 1. Least-squares superimposition of the most relevant active site residues of the wild-type Human CA I (His67) (dark structure) and the CA I M1 (Arg67) (light structure) molecule B, involved in sulfonamide inhibitor binding. Bound sulfanilamide is also shown.
140
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Fig. 1 shows the least-squares superimposition of the most relevant active site residues of the wild-type CA I (His67) (dark/blue structure) and the CA I M1 (Arg67) molecule B (bright/yellow structure) involved in sulfonamide inhibitor binding. In all cases the N1 nitrogen of sulfanilamide has displaced the zinc bound water/ hydroxide moiety and its O1 and O2 oxygens have taken the place of other solvent molecules present in the active site. The bond distances and angles of the catalytic zinc ion coordination polyhedron in the sulfanilamide /CA I M1 adduct are reported in Table 4. The noteworthy distances among sulfanilamide atoms and the atoms of the neighbouring residues are reported in Table 5. Significant changes are observed in the sulfanilamide molecule positioning, particularly regarding the p -amino group which is shifted away from residue Leu198, towards OE1 of Gln92 and consequently Arg67. This is possibly a result of the different conformation of the Gln92 side chain in CA I M1 with respect to the wild type isozyme (see Fig. 1). Comparing the wild type CA I amsulf adduct with molecule B of the CA I M1 /sulfanilamide adduct it appears that the sulfamido N1 /Zn(II) bond distance is ˚ . As a consequence, the van changed from 1.86 to 1.93A der Waals interaction between Thr199 OG1 and sulfa˚ ; and the mido O2 is shortened from 3.85 to 3.73 A hydrogen bond between Thr199 OG1 and sulfamido N1 ˚. is reduced from 3.25 to 2.84 A Therefore, in molecule B the p -amino moiety of the inhibitor is bent towards Arg67 due to the stronger interaction with Gln92 thus resulting in a longer bond distance between the metal ion and the N1 atom of the sulfamido group of sulfanilamide, partly counterbalanced by shorter hydrogen bonds with Thr199. The picture appears to be significantly different for molecule A (see Fig. 2), where the hydrogen bond interaction of sulfanilamide N2 with an exogenous ethylene glycol molecule, present in the crystallization buffer, seems to favour a stronger binding of the sulfamido group to the catalytic zinc ion and to Thr199 OG1. Indeed, in this case, the sulfamido N1 / ˚ (1.86 A ˚ in the wild type Zn(II) bond distance is 1.84 A CA I adduct). The van der Waals interaction between ˚ ) as well as the Thr199 OG1 and sulfamido O2 (3.40 A hydrogen bond between Thr199 OG1 and sulfamido N1 ˚ ) are also shorter than in molecule B (see Table (2.80 A 5). Since the inhibition constants were measured in the absence of ethylene glycol, we consider that the picture observed in molecule B is more consistent with such data. The overall differences observed between Molecule B and the wild type CA I, in particular the weaker bond of the inhibitor with the active metal ion could explain the reduced inhibition constants with aromatic sulfona-
mides observed for CA I M1 with respect to wild type CA I. We also examined the effect of the formation of the (Zn)2 adduct on sulfanilamide binding to CA I M1. The least-squares superimposition of the most relevant active site residues involved in sulfanilamide inhibitor binding to the CA I M1 (light/yellow structure) and its (Zn)2 adduct (dark/red structure) is reported in Fig. 3.
Fig. 2. Refined models of the sulfanilamide molecule and the interacting active site region for molecules A and B of CA1 M1 adducts.
˚) Distance X Zn (A CA I M1
His 94 No2 His 96 No2 His 119 Nd2 N1 (Sulfanilamide)
X Zn H94 CA I
Mol A
Mol B
1.93 2.01 2.01 1.84
1.91 2.03 2.01 1.93
CA I M1 Mol A
2.05 2.14 1.97 1.86
X Zn H96 CA I
Mol B
CA I M1
X Zn H119 CA I
Mol A
Mol B
102.1
101.9
105.4
CA I M1
X Zn N1 (sulfanilamide) CA I
Mol A
Mol B
116.3 95.3
114.3 96.5
113.3 96.9
CA I M1
CA I
Mol A
Mol B
114.0 111.6 114.9
117.6 110.4 113.2
108.1 118.4 114.3
M. Ferraroni et al. / Inorganica Chimica Acta 339 (2002) 135 /144
Table 4 Bond distances and angles of the catalytic Zn ion coordination polyhedron in the Sulfanilamide /CA I M1 adduct
141
142
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Fig. 3. Least-squares superimposition of the most relevant active site residues of the CA I M1 (light structure) and the CA I M1 (Zn)2 adduct (dark structure) molecule B, involved in sulfonamide inhibitor binding. Bound sulfanilamide is also shown. Table 5 Neighbors of Sulfanilamide Molecule in CA I, CA I M1, and CA I M1× Zn(II)2 adducts Sulfanilamide atom
N1 O1 O2 N2
Neighbor/atom
Zn(II) Thr199 OG1 Zn Thr199 OG1 Gln92 OE1 Gln92 NE2 Arg67 NH1 Arg67 NH2 EGL O2
Distances in CA I wild type
1.86 3.25 3.00 3.85 5.27 5.04
Distances in CA I M1
Distances in CA I M1 × Zn(II)2
Mol A
Mol B
Mol A
Mol B
1.84 2.80 3.07 3.40 4.59 6.47 7.42 5.43 3.11
1.93 2.84 3.07 3.73 4.23 6.37 7.37 5.45
1.74 2.97 3.35 3.51 3.38 5.32 4.45 4.23
1.93 2.65 2.97 3.64 3.42 5.62 3.43 3.10
Table 6 Bond Distances and Angles of both Zn Ion Coordination Polyhedrons in the Sulfanilamide-CA I M1× Zn(II)2 adduct
His 200 No2 His 64 No2 Arg 67 Wat241 O
His 94 No2 His 96 No2 His 119 Nd2 Sulfanilamide N1
˚) Distance X Zn (A
X Zn H200
X Zn H64
X Zn R67
X Zn Wat241 O
Mol A
Mol B
Mol A
Mol A
Mol B
Mol A
Mol B
Mol A
Mol B
1.92 2.25 1.91 2.23
2.18 2.20 2.08 2.11
104.6
104.2
114.8 116.0
118.6 105.9
97.4 114.1 108.5
106.1 112.9 109.3
1.87 2.10 2.03 1.74
2.01 1.97 2.00 1.93
Mol B
X Zn H96
X Zn 119
X Zn Sulfanilamide N1
Mol A
Mol B
Mol A
Mol B
Mol A
Mol B
94.2
97.4
113.4 100.0
109.0 103.1
118.3 117.9 110.9
115.0 119.1 111.6
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143
˚ ). The hydrogen bond between Thr199 OG1 and (3.42 A ˚ and with sulfanilamide N1 passes from 2.84 to 2.65 A ˚ . The sulfanilamide sulfanilamide O2 from 3.73 to 3.64 A N1 /Zn(II) bond distance is not further changed upon ˚ ). formation of the (Zn)2 adduct (1.93 A The tilting of the inhibitor molecule towards Gln92 and Arg67 is accompanied by small movements of Leu198 and His200 which appear to be further pushed in the opposite directions. The main differences observed in molecule A are the further shortening of the sulfanilamide N1 /Zn(II) bond ˚ ) counterbalanced by a longer Thr199 distance (1.74 A ˚ ) interaction. On the OG1 /sulfanilamide N1 (2.97 A opposite side of the inhibitor molecule the much longer distances between sulfanilamide N2 and Arg67 NH2 ˚ ) and NH1 (4.23 A ˚ ) are worthy of note. This is (4.45 A probably associated to the presence of a water molecule (Wat 179 in Fig. 4(A)) which has a strong van der Waals ˚ ). interaction with Arg67 (3.39 A Fluorescence experiments on the dansylamide (5(dimethylamino)naphthalene-1-sulfonamide) /CA I M1 adduct suggested that the presence of additional zinc ions induced the inhibitor molecule to move towards a more hydrophobic environment [32]. The present data show that, at least in the case of sulfanilamide, the presence of the second zinc induces a further shift of the inhibitor molecule towards the more hydrophilic site of the active site cavity, thus moving away from the hydrophobic region of Leu198. Dansylamide is certainly bulkier than sulfanilamide and therefore it is, at the moment, unfeasible to ascertain the possible orientational changes for such an inhibitor. Further experiments to investigate the inhibition of the CA I M1 (Zn)2 adduct by bulkier sulfonamides are currently in progress.
Acknowledgements Fig. 4. Refined models of the sulfanilamide molecule and the interacting active site region for molecules A and B of CA1 M1 (Zn)2 adducts.
The bond distances and angles of both zinc ion coordination polyhedrons in the sulfanilamide /CA I M1 (Zn)2 adduct are reported in Table 6. Upon binding of the second Zn(II) ion, the Arg67 side-chain moves towards the cavity, entering the second metal ion coordination sphere, and its rearrangement triggers a further repositioning of the inhibitor molecule. In molecule B, the p -amino moiety of the inhibitor establishes a new hydrogen bond with Arg67 NH2 (3.10 ˚ ) and a van der Waals interaction with Gln92 OE1 A
The authors of the present article want to acknowledge the valuable scientific contributions in bioinorganic chemistry of Helmut Sigel during his career, but also to thank him and his wife, Astrid, for the impressive editorial and organizational activity in favour of the bioinorganic community. We feel that a large part of our formation has been created along the years by the availability of fundamental book series and handbooks edited by them. The roles of Dr. R.E. Tashian and Dr. K.E. Wiebauer in the original discovery of the HCA I M1 variant and in the subsequent production of the cDNA clone, respectively, are gratefully acknowledged. We also wish to thank the Target Project on Biotechnologies CNR Italy.
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