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Originally published In Press as doi:10.1074/jbc.M310809200 on December 3, 2003

J. Biol. Chem., Vol. 279, Issue 8, 7223-7228, February 20, 2004
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Expression, Assay, and Structure of the Extracellular Domain of Murine Carbonic Anhydrase XIV

IMPLICATIONS FOR SELECTIVE INHIBITION OF MEMBRANE-ASSOCIATED ISOZYMES*

Douglas A. Whittingtons{ddagger}, Jeffrey H. Grubb§, Abdul Waheed§, Gul N. Shah§, William S. Sly§, and David W. Christianson{ddagger}||

From the {ddagger}Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the §Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104

Received for publication, October 1, 2003 , and in revised form, December 2, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Carbonic anhydrase (CA) XIV is the most recently identified mammalian carbonic anhydrase isozyme, and its presence has been demonstrated in a number of tissues. Full-length CA XIV is a transmembrane protein composed of an extracellular catalytic domain, a single transmembrane helix, and a short intracellular polypeptide segment. The amino acid sequence identity of human CA XIV relative to the other membrane-associated isozymes (CA IV, CA IX, and CA XII) is 34-46%. We report here the expression and purification of both the full-length enzyme and a truncated, secretory form of murine CA XIV. Both forms of this isozyme are highly active, and both show an abrogation of activity in the presence of 0.2% SDS, in contrast to the behavior of murine CA IV. We also report the crystal structure of the extracellular domain of murine CA XIV at 2.8 Å resolution and of an enzyme-acetazolamide complex at 2.9 Å resolution. The structure shows a monomeric glycoprotein with a topology similar to that of other mammalian CA isozymes. Based on the x-ray crystallographic results, we compare and contrast known structures of membrane-associated CA isozymes to rationalize the structural elements responsible for the SDS resistance of CA IV and to discuss prospects for the design of selective inhibitors of membrane-associated CA isozymes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Carbonic anhydrases (CAs)1 are zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide (). This simple chemical reaction has important implications for pH homeostasis, carbon dioxide and ion transport, respiration, and many other critical processes in living systems (1). The CAs fall into three distinct classes ({alpha}, {beta}, and {gamma}) on the basis of sequence and structural similarity (2). All mammalian CAs belong to the {alpha} class, of which there are at least 11 enzymatically active isozymes. Additional CA-related proteins lacking intact zinc-binding sites have been identified based on amino acid sequence identity to active isozymes (3). The CAs are ubiquitous in mammalian tissues, but individual isozymes display tissue-specific distributions (1). Further distinctions in isozyme localization are due to the cytosolic, membrane-associated, or secretory nature of specific CAs. The varying tissue distributions have been exploited for the development of CA inhibitors targeted to specific regions of the body, the most notable example being topically applied compounds such as dorzolamide and brinzolamide for the treatment of glaucoma (4). Subtle structural differences among the CA isozymes also hold promise for the development of isozyme-specific inhibitors, certain examples of which have been demonstrated (5).

The most recently identified mammalian CA isozyme is CA XIV. Through the use of Northern blotting and reverse transcriptase-polymerase chain reaction techniques, CA XIV mRNA has been demonstrated in kidney, liver, brain, skeletal muscle, heart, and lung (6-8). The protein itself has been identified in murine and human brain, murine liver, and rat and murine kidney (8-10). CA XIV is a bitopic membrane protein with an extracellular N-terminal catalytic domain, a single membrane-spanning segment, and a small intracellular C-terminal polypeptide containing potential phosphorylation sites (6, 7). The first 15 amino acids are hydrophobic and constitute a signal sequence, and the catalytic domain contains one putative N-glycosylation site (6, 7). This topology is similar to that of the other transmembrane isozymes, CA IX and CA XII. A fourth isozyme, CA IV, is also membrane-associated, but the post-translational attachment of a glycosylphosphatidylinositol group to the C terminus of CA IV serves as the membrane anchor rather than the polypeptide itself (11). The amino acid sequence identity of human CA XIV relative to the other three membrane-bound CA isozymes is 34-46%. CA XIV also shares 38% sequence identity with CA VI, an extracellular, secreted isozyme found in saliva.

Despite similarities in amino acid sequences and overall topology, the membrane-associated CAs differ in tissue distribution. CA XIV is found in regions of liver cells distinct from the location of CA IV (10), although certain regions of the kidney show positive immunostaining for both of these isozymes, suggesting redundant function (8). Intriguingly, the presence of an extracellular carbonic anhydrase has long been suspected in mammalian brain (12, 13). Known CA inhibitors, including compounds that are impermeable to cells, were shown to enhance the extracellular alkaline shift observed in slices of hippocampus after synaptic transmission (12-14). Recent immunostaining results identify CA XIV on neurons and axons in both mouse and human brain, suggesting that this isozyme is responsible for modulating pH shifts during excitatory synaptic transmission (9). The other two transmembrane isozymes, CA IX and CA XII, show a varied tissue distribution, but both are overexpressed in certain cancers, and their transcription is regulated by the von Hippel-Landau tumor suppressor (15-22).

We report here the expression, purification, and assay of the soluble, extracellular domain of murine CA XIV and its structure determination by x-ray crystallographic methods. The x-ray structure confirms that CA XIV is a glycoprotein and helps define its quaternary structure relative to its solution behavior and its similarity to the related CA XII isozyme. The structure of murine CA XIV complexed with acetazolamide is also presented. Based on the structures of CAs IV, XII, and XIV, we rationalize the structural elements responsible for the unique SDS resistance of CA IV. This resistance allowed CA IV to be solubilized from tissues by SDS and purified in the presence of SDS, conditions under which other known CAs were inactive (11); subsequently, resistance to SDS became a practical means of determining the contribution of CA IV to total activity in tissues. Finally, prospects for the design of inhibitors selective for the extracellular CA isozymes are discussed based on the structural data.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Expression and Purification—The cDNA was cloned by PCR using mRNA from C57BL6 mouse kidney using primers designed by Mori et al. (6). The sequence2 differs from that reported for CA XIV from BalbC (6) and predicts a His instead of a Gln at residue 108 (see Fig. 3) and a 3-base pair in-frame deletion of Ala-263. Mammalian expression vectors containing the cDNA of the wild-type, full-length membrane form and secretory form (I261X) of murine CA XIV were constructed as described (9, 23). Stable Chinese hamster ovary clones expressing the secretory or the full-length membrane form of murine CA XIV were isolated and characterized by CA activity following established procedures (23). A full-length, membrane form of murine CA XII cDNA was subcloned into the mammalian expression vector pCXN (24) and transiently expressed in COS-7 cells (19). The enzyme expression was analyzed by CA activity measurements (25).



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  		FIG. 3.
Structure-based sequence alignment of human CA II (Protein Data Bank code 2CBA [PDB] ; Ref. 50), human CA IV (Protein Data Bank code 1ZNC [PDB] ; Ref. 41), murine CA IV (Protein Data Bank code 2ZNC [PDB] ; Ref. 42), human CA XII (Protein Data Bank code 1JCZ [PDB] ; Ref. 31), and murine CA XIV. Human CAs XIV and IX are also included for comparison. Amino acid numbering corresponds to human CA II. Absolutely conserved residues are colored red, N-glycosylation sites are green, and diamonds denote zinc ligands. Secondary structure elements for murine CA XIV are depicted schematically below the sequence. Putative transmembrane segments are indicated by a gray box.

 
The I261X secretory form of murine CA XIV, designated CA14x, was purified from secretion medium using CA inhibitor affinity chromatography (11). The secretion medium was applied to a CA inhibitor affinity column equilibrated with 10 mM HEPES (pH 7.5). The unbound protein was removed by washing with equilibration buffer containing 0 mM and then 150 mM NaCl. The bound enzyme was eluted with 0.1 M sodium acetate containing 0.5 M sodium perchlorate (pH 5.5). The eluted enzyme was concentrated and dialyzed against 10 mM Tris-SO4 (pH 7.5). The homogeneity of the enzyme was assessed by size exclusion chromatography using Sephacryl S-300, SDS-PAGE, and specific activity (25).

Activity Assays—CA activity was measured by the procedure of Maren (26), as described (25). SDS-resistant CA activity was determined on affinity pure CA samples or membrane-bound CA samples preincubated with 0.2% SDS at room temperature for 30 min prior to activity measurements. The protein concentration was determined by the micro Lowry procedure (27). CA activity is expressed in enzyme units/mg of cell protein for unpurified enzyme or in enzyme units/mg of affinity pure CA.

Crystallization and Data Collection—The CA14x protein was crystallized at room temperature by the hanging drop vapor diffusion method. Drops containing 1.8 µl of 7 mg/ml enzyme in 20 mM sodium phosphate (pH 7.2) and 150 mM NaCl were mixed with 1.8 µl of precipitant buffer (5.5% (w/v) polyethylene glycol 4000, 0.1 M sodium acetate, pH 4.8, 20 mM NaCl) and equilibrated over a well containing 1.0 ml of precipitant buffer. Crystals appeared in the drops within 48 h and grew as long, thin rods to maximum dimensions of 0.7 x 0.03 x 0.03 mm3. For data collection, a microspatula was used to break the rods into shorter pieces that were subsequently harvested into a stabilizing buffer containing 10% (w/v) polyethylene glycol 4000, 0.1 M sodium acetate (pH 4.8), 20 mM NaCl, and 10% (v/v) glycerol. After sequential transfers to stabilizing solutions containing 15 and 25% (v/v) glycerol, the crystals were flash cooled in liquid nitrogen. Diffraction data to 2.8 Å resolution were collected from a single CA14x crystal at beamline X25 of the National Synchrotron Light Source at the Brookhaven National Laboratories. The data were processed with the HKL suite (28) and TRUNCATE (29). The crystals belonged to space group P21 with unit cell dimensions a = 59.0 Å, b = 75.6 Å, c = 73.2 Å, and {beta} = 98.9°; with two molecules in the asymmetric unit, the Matthew's coefficient VM = 2.65 Å3/Da (53% solvent content). For preparation of the CA14x-acetazolamide complex, the crystals were soaked in a stabilizing solution containing 10% (w/v) polyethylene glycol 4000, 22% (v/v) glycerol, 0.1 M sodium acetate (pH 5.5), 20 mM NaCl, and 5 mM acetazolamide for 90 h prior to flash cooling in liquid nitrogen. The data were collected at beamline X12C of National Synchrotron Light Source and processed as described. The data collection statistics are recorded in Table I.


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  		TABLE I
Data collection and refinement statistics

 
Structure Determination and Refinement—The molecular replacement calculations were performed with AMoRe (30) using the atomic coordinates of CA XII (Protein Data Bank code 1JCZ [PDB] ) as a search probe (31). Rotation searches using diffraction data from 15-3.5 Å resolution yielded two clear solutions, as ranked by correlation coefficient. Subsequent translation searches placed two molecules in the asymmetric unit, and rigid body refinement lowered the R factor to 0.453. Model building was performed with the program O (32), and simulated annealing, conjugate gradient positional refinement, and temperature factor refinements were performed with CNS (33). Strict noncrystallographic symmetry constraints were applied during initial refinement cycles and were subsequently released into heavily weighted restraints ({omega} = 300 kcal/mol Å2) for all atoms except those that displayed obvious differences between the two molecules, e.g. due to crystal lattice contacts. The data were refined against a maximum likelihood target function as implemented in CNS, and a bulk solvent correction was employed (ksol = 0.35 e Å-3 was defined) (34). Automatic B factor corrections were not used. Solvent molecules were built into the model at positions where the Fo-Fc maps contained peaks of >=3.0 {sigma} that displayed appropriate hydrogen bonding interactions. The final model had Rcryst = 0.234 (Rfree = 0.274) and included two copies of the CA14x polypeptide, two zinc ions, two acetate ions, four N-acetylglucosamine rings, one mannose ring, and 90 solvent molecules. Geometric parameters were analyzed with PROCHECK (35); a total of 85 and 14% of the backbone {phi}-{psi} conformations adopt most favorable and additionally allowed conformations, respectively.

The structure of the CA14x-acetazolamide complex was solved using the difference Fourier method starting from the wild-type CA14x structure less all zinc ions, acetate ions, solvent molecules, and sugar moieties. Rigid body, positional, and grouped temperature factor refinements in CNS resulted in a final model having Rcryst = 0.207 (Rfree = 0.253). The refinement protocol was the same as that used for the native CA14x structure, except that an initial B factor correction was applied to the data. The final model contained two CA14x polypeptide chains, two zinc ions, two acetazolamide molecules, four N-acetylglucosamine rings, two mannose rings, and 32 water molecules. The data refinement statistics for both structures are recorded in Table I.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Molecular Characterization and Enzyme Activity—Affinity-purified CA14x migrates as a 44-kDa polypeptide on SDS-PAGE (Fig. 1). However, the calculated molecular mass of CA14x deduced from its amino acid sequence is 29.5 kDa. Therefore, the increase in apparent molecular mass of CA14x and the presence of one consensus sequence for N-glycosylation suggest that murine CA XIV is a glycoprotein. Accordingly, the affinity-purified mouse CA14x produced in the glycosylation-defective Lec-1 cell line shows a slightly smaller apparent molecular mass on SDS-PAGE (data not shown). Moreover, CA14x eluted as single peak of 120-kDa mass during size exclusion chromatography on Sephacryl S-300. Because the mass of the monomeric glycopeptide estimated by SDS-PAGE was 44 kDa, these results suggest either that native CA14x exists as a multimer in solution or that its migration on an S-300 column deviates from that of an ideal globular protein. X-ray crystallographic results support the latter explanation (see below).



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  		FIG. 1.
Size exclusion chromatography of the secretory form of murine CA XIV. A, affinity-purified CA14x was applied to a calibrated Sephacryl S-300 column that was equilibrated in 10 mM HEPES (pH 7.5), 150 mM NaCl buffer. The column was eluted in 3.5-ml fractions that were assayed for enzyme activity, enzyme units/fraction x 10-3 (•) and monitored at 280 nm ({blacksquare}) for protein. B, fractions containing protein were analyzed by SDS-PAGE, and the polypeptides were visualized by silver staining. The arrow indicates the apparent molecular mass (kDa) of murine CA14x.

 
The specific activity of pure recombinant glycosylated CA14x produced in Chinese hamster ovary cells was compared with unglycosylated murine CA II and glycosylated murine CAs IV and XII. The results are presented in Table II. The specific activity of CA XIV (3284 enzyme units/mg) is higher than any of the other isozymes investigated, including CA II. Unlike murine CA IV, CA14x and the secretory form of CA XII were SDS-sensitive. Membrane-associated CA XII and CA XIV were also SDS-sensitive, indicating that CA IV is more stable than even the wild-type transmembrane isozymes. Because CA IV, CA XII, and CA XIV share a common disulfide bond between Cys-23 and Cys-203, the increased stability of CA IV must be ascribed to the additional disulfide bond between Cys-6 and Cys-13 of this isozyme.


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  		TABLE II
Specific activity and SDS resistance of murine carbonic anhydrase isozymes

 
Overall Structure of Murine CA XIV—The structure of the extracellular catalytic domain of murine CA XIV reveals a polypeptide fold characteristic of the {alpha}-CA isozymes in which a 10-stranded {beta}-sheet forms the core of the molecule (Fig. 2). A single disulfide linkage is present between residues Cys-23 and Cys-203 that is identical to disulfide bonds found in the membrane-associated isozymes CA IV and CA XII. This disulfide bond helps to stabilize a polypeptide loop in the active site containing Thr-199, a residue that promotes efficient catalysis by orienting the nucleophilic zinc-bound solvent molecule through a hydrogen bonding interaction (36, 37). Additionally, because CA14x was produced in Chinese hamster ovary cells, the molecule is glycosylated. Both molecules in the asymmetric unit exhibit electron density consistent with N-glycosylation of Asn-195. The CA14x-acetazolamide structure exhibits the highest quality electron density for the carbohydrate. In molecule B, four of the sugar rings that form the core pentasaccharide commonly found in N-glycosylation are visible: two N-acetylglucosamine and two mannose moieties. Molecule A exhibits electron density for only the two N-acetylglucosamines.



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  		FIG. 2.
Structure of the murine CA XIV extracellular domain positioned over the cell membrane. The catalytic zinc ion is indicated by an orange sphere. The disulfide bond between Cys-23 and Cys-203 and the N-linked saccharide at Asn-195 are shown in ball-and-stick representation.

 
The catalytic domain of CA XIV exhibits only minor differences compared with the structure of CA XII (31), the most closely related transmembrane isozyme. Two loop regions vary between these isozymes; CA XIV has an insert in the Gly-151-Glu-153 loop and a deletion in the Thr-233-Pro-240 loop relative to CA XII. A structure-based sequence alignment is presented in Fig. 3. The catalytic domains of CA XII and CA XIV contain N-glycosylation sites at differing locations in their sequence, but the protein backbones still superimpose well (root mean square deviation = 1.1 Å for 255 C{alpha} atoms). The most striking difference between CA XII and CA XIV is quaternary structure; CA XII is a dimer with 2200 Å2 buried surface area between monomers, whereas CA XIV appears monomeric. The largest surface area buried between adjacent CA XIV molecules in the crystal lattice is 730 Å2 (365 Å2/monomer), which includes the surface area of the carbohydrate, whereas statistical analyses of the buried surface between a large sampling of biological dimers suggests a minimum buried surface area of 1700 Å2 (860 Å2/monomer) (38). The packing of these two CA14x molecules also occludes the active site of one, further arguing against the existence of a functional CA XIV dimer, and the majority of amino acids present in the human CA XII dimer interface are not conserved in human or murine CA XIV. Even so, the proposed transmembrane {alpha}-helix of full-length CA XIV contains one GXXXG sequence motif, a feature that has been shown to promote helix-helix interactions in membranes (39, 40). The CA XII transmembrane sequence contains a similar motif that is proposed to facilitate dimerization (31). Although the crystal structure of CA14x presents no evidence for a dimeric protein, we cannot exclude the possibility that the transmembrane {alpha}-helix of CA XIV promotes homodimerization or heterodimerization of the full-length enzyme in the membrane.

Two other membrane-associated isozymes exist with similarity to CA XIV, namely CA IV and CA IX. The structure of the extracellular catalytic domain of CA IX is not known, but crystal structures of human and murine CA IV have been reported (41, 42). Comparing the structures of CAs IV and XIV reveals an overall similarity of the {beta}-sheet superstructure but highlights two regions of notable difference. First, the N terminus of CA IV has an insert of 5 amino acids relative to other {alpha}-CAs. This insert contains an additional, short {alpha}-helix that is not present in the CA XII or XIV structures. As a result of this extra sequence, the N-terminal portion of the CA IV structure makes van der Waals' contact with the Leu-230-Gln-238 loop region of the molecule. Such interactions are absent in other CA structures (Fig. 4). CA IV also contains an extra disulfide bond within this small insert between Cys-6 and Cys-13. This extra disulfide bond and the contact between the N-terminal insert and the Leu-230-Gln-238 loop appear to stabilize this isozyme against inactivation by SDS, resistance to which is unique to CA IV. The second notable difference between the CA IV and CA XIV structures is found in the loop region from Ser-125 to Gly-140. In CA XIV and in other CA isozymes, this region contains a short {alpha}-helix that flanks the top edge of the active site cleft; however, in CA IV this same region exists as an extended loop exhibiting substantial disorder (41, 42). Indeed, the difference in this loop region has been suggested to account for differences in inhibitor Ki values between human CAs II and IV (42, 43).



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  		FIG. 4.
N-terminal insert of human CA IV relative to human CA XII and murine CA XIV. The CA IV sequence is depicted in red with the side chains labeled, and the CA XII and CA XIV sequences are shown as C{alpha} traces in blue and green, respectively. The additional intramolecular contacts in CA IV between the N-terminal insert and the 130's loop combined with the additional disulfide bond between Cys-6 and Cys-13 (depicted in gold) stabilize CA IV in the presence of SDS. Inserts in the CA IV sequence are numbered according to the point of insertion with a letter appended (e.g. 11A, 11B, etc.).

 
Active Site Structure—As in other CA isozyme structures, the catalytic zinc ion is located at the bottom of a conical active site cleft where it is ligated by His-94, His-96, and His-119. The trigonal bipyramidal zinc coordination polyhedron is completed by a solvent molecule and an acetate ion from the crystallization buffer (Fig. 5A). Thr-199 accepts a hydrogen bond from the zinc-bound solvent molecule (presumably water at pH 4.8). One acetate oxygen atom coordinates to zinc, whereas the second acetate oxygen atom accepts hydrogen bonds from the zinc-bound solvent molecule and the backbone NH group of Thr-199. This arrangement is analogous to the binding of acetate to CA II and CA XII (31, 44), and it mimics the binding of the bicarbonate ion product (37, 45).



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  		FIG. 5.
A, the active site of murine CA14x in which simulated annealing omit electron density maps indicate the position of the catalytic zinc ion (orange, 10 {sigma} contour) and the zinc ligands (turquoise, 3.0 {sigma} contour). Hydrogen bonds are indicated as red dashed lines. The zinc-ligand distances are as follows: Zn2+-His-94, 2.0 Å; Zn2+-His-96, 2.3 Å; Zn2+-His-119, 2.1 Å; Zn2+-OH2, 2.2 Å; Zn2+-acetate, 2.1 Å. B, simulated annealing omit electron density map depicting the active site of the CA14x-acetazolamide complex with the inhibitor omitted from the final model (magenta,3{sigma} contour). Hydrogen bonds are indicated as red dashed lines. C, schematic diagram of acetazolamide binding to murine CA XIV.

 
Amino acids in the immediate environment of the catalytic zinc ion are conserved across all active {alpha}-isozymes, and CA XIV is no exception. CA XIV also contains the proton shuttle residue His-64, which is present in all enzymes with high activity. Because proton release is the rate-limiting step in catalysis (46, 47), isozymes without this residue display substantially lower activity (48). In CA XIV, His-64 adopts the "out" conformer (data not shown). This orientation is typically observed in CA structures solved at low pH (31, 41, 49). Because of the moderate 2.8 Å resolution of the CA XIV structure, no ordered solvent network is observed between His-64 and the zinc-bound solvent molecule. Such a solvent network was observed in the 1.54-Å resolution structure of CA II (50).

Inhibitor Binding to CA XIV—Structural similarity between CA XIV and other isozymes in the active site cleft adjacent to the catalytic zinc ion suggests that aromatic sulfonamides such as acetazolamide, a potent class of CA inhibitors, will bind tightly to CA XIV. Notably, 1 µM acetazolamide reduces CA XIV activity in extracts from COS-7 cells by 84% (7). Acetazolamide and a membrane-impermeable CA inhibitor, benzolamide, enhance the extracellular pH shifts in neurons (12, 14), in which immunostaining has recently demonstrated the presence of CA XIV (9). To examine the binding of acetazolamide to murine CA XIV, we determined the crystal structure of the CA14x-acetazolamide complex at 2.9-Å resolution. Following x-ray crystallographic refinement and model building of the CA14x polypeptide, zinc ions, and carbohydrate, a clear, continuous feature of electron density was present in the active site in difference Fourier maps (Fig. 5B). As with acetazolamide binding to other isozymes, the ionized sulfonamide NH- group coordinated to the zinc ion and donated a hydrogen bond to Thr-199. Additional hydrogen bond contacts were made between the backbone nitrogen atom of Thr-199 and a sulfonamide O atom and between the hydroxyl group of Thr-200 and a nitrogen atom on the 1,3,4-thiadiazole ring. The contacts made between acetazolamide and the CA XIV active site are depicted schematically in Fig. 5C.

Among the extracellular CA isozymes, unique variations in CA XIV occur at several positions near the top of the active site cleft (Fig. 6). This region lies adjacent to the end of the acetazolamide inhibitor in the CA XIV active site, and these differences could potentially be exploited in the design of isozyme-specific inhibitors. Notably, the residues lining the murine CA XIV active site cleft are identical to those in human CA XIV, allowing us to draw valid inferences about the human isozyme from the murine structure. In CA XIV, Tyr-204 is found in place of Asp or Asn residues in the human CA IV and XII sequences, a difference that eliminates a potential hydrogen bond interaction on one face of the active site cleft. Gln-67 from CA XIV replaces either Lys-67 or Met-67 from CA XII or CA IV, respectively, providing another unique feature to the cleft surface. Finally, the combination of Ala-91 and Leu-131 creates a hydrophobic patch in CA XIV where Thr-91 and Ala-131 are found in CA XII. CA IV also differs in this region because of the disorder of the "130's segment," which is found as an ordered {alpha}-helix in CA XII, CA XIV, and other isozymes.



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  		FIG. 6.
Comparison of the contours of the active site clefts in human CA IV and the extracellular domains of human CA XII and murine CA XIV. The zinc ion is indicated by an orange sphere, and selected residues are depicted. Residues in all of the isozymes are numbered based on alignment with human CA II.

 
Potent inhibitors of CA isozymes bind to the catalytic zinc ion. Because of the absolute identity of amino acids adjacent to the catalytic metal ion in membrane-associated CA IV and transmembrane CA XII and CA XIV, identical intermolecular interactions are expected between the protein and the zinc-binding group for inhibitors of these enzymes. To achieve isozyme specificity, potential CA inhibitors should exhibit differences in regions removed from the zinc-binding head group that exploit the variations in the active site cleft discussed above. Compounds that bind with higher affinity to CA II versus CA IV exhibit just such differential interactions with the active site clefts in those enzymes (43). The extracellular placement of the membrane-associated CA active sites also facilitates their selective targeting by charged and/or membrane-impermeable compounds. This combination of extracellular active sites, subtle differences in molecular structure of the active site cleft, and tissue-specific expression profiles for the membrane-associated CA isozymes holds promise for their selective inhibition by small molecules.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 1RJ5 and 1RJ6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by National Institutes of Health Grants GM45614 (to D. W. C.) and DK40163 (to W. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence may be addressed. Tel.: 314-977-9201; Fax: 314-776-1183; E-mail: slyws{at}slu.edu.

|| To whom correspondence may be addressed. Tel.: 215-898-5714; Fax: 215-573-2201; E-mail: chris{at}xtal.chem.upenn.edu.

1 The abbreviation used is: CA, carbonic anhydrase. Back

2 In this work, the CA XIV sequence was numbered based on alignment with human CA II (see Fig. 3). Inserted residues are numbered by the point of insertion with a letter appended (e.g. 11A, 11B, etc.). Back


   ACKNOWLEDGMENTS
 
We thank the National Synchrotron Light Source (Brookhaven National Laboratories) for beamline access and Drs. J. David Cox and Michael Rynkiewicz for assistance with data collection.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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