Biochemical Analysis of the Modular Enzyme Inosine 5′-Monophosphate Dehydrogenase

Protein Expression and Purification 17, 282–289 (1999) Article ID prep.1999.1136, available online at http://www.idealibrary.com on

Biochemical Analysis of the Modular Enzyme Inosine 59-Monophosphate Dehydrogenase Elmar Nimmesgern, 1 James Black, Olga Futer, John R. Fulghum, Stephen P. Chambers, Christopher L. Brummel, Scott A. Raybuck, and Michael D. Sintchak Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, Massachusetts 02139-4242

Received June 16, 1999, and in revised form August 9, 1999

Two prominent domains have been identified in the X-ray crystal structure of inosine-5*-monophosphate dehydrogenase (IMPDH), a core domain consisting of an a/b barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1–108 and 244 –514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99 – 244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% a-helix and 30% b-sheet vs 33% a-helix and 29% b-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wildtype and core domain proteins by its content of secondary structure, with only 15% each of a-helix and b-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs. © 1999 Academic Press

Inosine 59-monophosphate dehydrogenase (IMPDH) 2 is a key enzyme in the de novo biosynthesis of guanine nucleotides. IMPDH (E.C. 1.1.1.205) catalyzes the NAD-dependent conversion of IMP to XMP. The enzyme is a tetramer formed by subunits of about 55 kDa molecular weight. In many species two isoforms (type I and type II) have been described (in man 514 amino acids each with 84% sequence identity (1)). Using specific inhibitors of IMPDH it has been shown that the proximal biochemical effect of blocking the enzyme in sensitive cell types—those that rely on the de novo as opposed to the salvage pathway to provide guanine nucleotides—is a decrease in cellular guanine nucleotide levels, which in turn blocks RNA and DNA synthesis as well as the glycosylation of membrane proteins. Via this mechanism, IMPDH inhibition has reversible antiproliferative, antiviral, antiparasitic, and immunosuppressive effects (2). The immunosuppressive effect of IMPDH inhibitors has been useful in the clinic because lymphocytes are especially dependent on the de novo pathway. An ester prodrug of the IMPDH inhibitor mycophenolic acid (MPA), mycophenolate mofetil (CellCept), is approved for the prevention of rejection after kidney and heart transplantation. Inhibition of IMPDH may also explain the antiviral effect of ribavirin. Ribavirin is a nucleoside and its 59-monophosphate is a competitive IMPDH inhibitor (3). Ribavirin is approved in the United States as monotherapy (Virazole) for the treatment of respiratory syncytial virus and in combination with interferon-a (Rebetron) for the treatment of hepatitis C. The X-ray crystal structure of hamster IMPDH (II) in complex with MPA has been solved (4). IMPDH consists of two domains, an a/b barrel core domain which contains the active site and an inserted subdo-

1 To whom correspondence should be addressed. Fax: (617) 577 6400. E-mail: [email protected].

2 Abbreviations used: IMPDH, Inosine 59-monophosphate dehydrogenase; MPA, mycophenolic acid; CBS, cystathione-b-synthase.

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BIOCHEMICAL ANALYSIS OF IMPDH

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main including residues 110 –244 that is less well defined in the crystal structure. In order to define more precisely the function of the two domains of IMPDH, we have separately expressed and purified the core domain and the subdomain as well as human (II) wild-type enzyme. Based on the structural information of the hamster (II) wild-type IMPDH, we designed a core domain construct in which the subdomain is excised and replaced with a 4-aminoacid linker. This creates a construct representing 74% of the amino acids of the full-length enzyme. The subdomain containing the remaining 26% of the amino acids of full-length IMPDH was expressed as a T7- and His-tagged fusion protein to facilitate purification. The results presented in this paper describe a catalytically active tetrameric core domain of IMPDH. In contrast, the subdomain is a monomer or a dimer and enzymatically inactive. CD analysis reveals differences in the secondary structure content of the two domains of IMPDH. These results will aid in a more detailed structural and functional characterization of human IMPDH.

AAC-39) and 3 (59-CGGGGTACCCATATGGCCGACTACCTGATTAGTGGGGGCACGTCC-39) the sequence coding for the N-terminus of IMPDH including amino acid 108 was amplified. The sequence coding for the C-terminal portion of the core domain protein starting at amino acid 245 extending to the BspM1 site was amplified using primers 2 (59-AAAGTGAAGATGCGTACTGGTCTGTGTGGGGCAGCC-39) and 4 (59-TTTGTCCTGCACAGCACC-39). Primers 1 and 2 partially overlap and create a 4-amino-acid linker (Ile-Arg-ThrGly) replacing the IMPDH subdomain. The two generated PCR products were used as the template in a third reaction with primers 3 and 4. The PCR-generated fragment was digested with NdeI/BspMI, cloned into unique sites within human IMPDH (II) cDNA, and sequenced in its entirety. The core domain IMPDH construct was then subcloned into the pSPC27 vector for expression under IPTG-inducible Tac promotor, as described above. pSPC27 is a high copy number derivative of pFlag1 (7).

MATERIALS AND METHODS

A 437-bp EcoRI–PvuII fragment, containing the IMPDH (II) subdomain, was isolated from human IMPDH (II) cDNA. The fragment was blunt-ended with T4 polymerase and cloned into the PvuII site of pRSETb (Invitrogen). Recombinants were selected with the correct orientation of the IMPDH (II) subdomain within the vector. A stop codon was introduced, by site-directed mutagenesis, at the 39 end of the IMPDH sequence to terminate the open reading frame. The resulting vector pRSETb-HIS 6-IMPDH (II) subdomain contained the IMPDH (II) subdomain (residues 99 – 244) with enterokinase cleavable T7 and HIS 6 tags at its N-terminus. To prepare HIS 6-IMPDH (II) subdomain from bacterial cells, competent E. coli cells BL21 (DE3) (Novagen) freshly transformed with pRSETb-HIS 6-IMPDH (II) subdomain were grown in complex media supplemented with ampicillin (100 mg/ml), in a 10-liter fermentor at 37°C. Once the cell density reached an OD 600 of 0.9 the temperature of the culture was rapidly reduced to 30°C and induced with 1 mM IPTG. The cells were harvested 2 h postinduction and flash frozen at 270°C prior to purification.

Expression of Human IMPDH (II) in Escherichia coli Human IMPDH (II) cDNA was isolated from a human spleen cDNA library (Clontech). A 1545-bp fragment was produced by PCR amplification using cDNA primers (59-ATGGCCGACTACCTGATTAGTGGGGGCACGTCCTACGTGCCAGAC-39 and 59-TCAGAAAAGCCGCTTCTCATACGAATGGAGGCTATGGACGCCACC-39) corresponding to the N- and C-termini, respectively, of the human IMPDH (II) sequence (1). Subsequently, a NdeI site (CATATG) was introduced at the AUG translation initiation codon of the IMPDH (II) gene, and a XbaI site distal to its 39 termination. The IMPDH gene was cloned as a NdeI–XbaI fragment into the expression vector pFlag I (IBI), producing a vector designated phIMPDH (II). Human IMPDH (II) was expressed in H712 (gua 2), a mutant E. coli strain deficient in IMPDH (5), as described (6). Cells were grown at 37°C in a 10-liter fermentor (B. Braun), on complex media supplemented with 1 mM IPTG, allowing constitutive expression. Cells were harvested after 14 h, flash frozen, and stored at 270°C prior to purification.

Expression of Human HIS 6-IMPDH (II) Subdomain in E. coli

Protein Purification Expression of Human IMPDH (II) Core Domain Protein in E. coli Using the human IMPDH (II) cDNA, described above, as the template, a DNA construct of the human IMPDH (II) core domain was prepared in a three-step PCR method. In the first PCR, using primers 1 (59CCCACACAGACCAGTACGCATCTTCACTTTCCG-

All purification steps were performed on ice or at 4°C unless described otherwise. The human IMPDH (II) protein was purified as described for hamster IMPDH (II) (6). Briefly, 29 g of cell paste was homogenized in 180 ml of buffer A (300 mM KCl, 50 mM Tris/HCl, pH 8, 2 mM EDTA, 1.5 M urea, 10 mM b-mercaptoethanol) containing 0.2 mM PMSF

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and 1 mg/ml each of pepstatin, leupeptin A, and E64 (inhibitors from Boehringer Mannheim) and the cell suspension passed twice through a microfluidizer (Microfluidics Corp.). A soluble fraction was prepared by 45 min centrifugation at 54,000g. IMPDH was precipitated from the supernatant by addition of ammonium sulfate to 25% (w/v) and the precipitate was collected by centrifugation as above. The ammonium sulfate precipitate was dissolved in 200 ml buffer B (300 mM KCl, 50 mM Tris/HCl, pH 8, 2 mM EDTA, 1.5 M urea, 10 mM b-mercaptoethanol) and loaded onto a 250-ml IMP-Sepharose column (prepared by coupling IMP to epoxy-activated Sepharose as described (6)) equilibrated in the same buffer. The column was washed, and bound IMPDH eluted with 2 mM IMP in buffer B. IMPDH-containing fractions were pooled, concentrated to 40 ml, and loaded onto an 1800 ml Sephacryl S-300 column (Pharmacia) equilibrated in buffer B. Elution fractions containing tetrameric IMDPH were pooled, concentrated to a protein concentration of about 2 mg/ml, and frozen at 270°C. The core domain protein was purified with a method similar to that for the wild-type protein with some modifications. Cell paste (24 g) was homogenized in 140 ml buffer C (as buffer A but 100 mM KCl) containing inhibitors as above. A soluble extract was prepared as described above and the core domain protein precipitated from the supernatant by addition of 10% (w/v) ammonium sulfate. The precipitate was collected by centrifugation as above. The ammonium sulfate precipitate was dissolved in 45 ml buffer C and loaded onto a 50-ml IMP-Sepharose column equilibrated in the same buffer. After loading, the column was washed with buffer D (as buffer B but 100 mM KCl) and bound IMPDH was eluted with 2 mM IMP in buffer D. Fractions containing active enzyme were pooled and concentrated in a stirred ultrafiltration device at room temperature to a protein concentration of 7.5 mg/ml and frozen and stored at 270°C. For purification of the subdomain protein, 30 g of cell paste was homogenized in 180 ml of buffer E (300 mM KCl, 50 mM Tris/HCl, pH 8.0, 1.5 M urea, 0.5% Chaps) containing 10 mM b-mercaptoethanol (Sigma) and inhibitors as above. The homogenized cell paste was passed twice through a microfluidizer and the crude extract was centrifuged for 45 min in the SS34 rotor at 43,000g. The supernatant was diluted with an equal volume of buffer E. The soluble extract was loaded onto a 15-ml Talon (Clontech) column equilibrated in buffer E with 5 mM b-mercaptoethanol. The column was washed with 30 ml of the same buffer, 30 ml buffer F (50 mM Tris HCl, pH 8.0, 300 mM KCl, 10% glycerol, 0.5% Chaps, 5 mM b-mercaptoethanol) as well as 30 ml buffer F containing 5 mM imidazole. The His-tagged subdomain protein was eluted with 100 mM imidazole in buffer F. Fractions containing the subdomain pro-

tein were pooled and DTT was added to a final concentration of 20 mM. Protein was dialyzed against buffer G (as buffer F but 50 mM instead of 300 mM KCl and 20 mM DTT instead of 5 mM b-mercaptoethanol) and then loaded onto a MonoQ HR 10/10 column (Pharmacia, 6.6 ml) equilibrated in buffer G. After loading the protein, the column was washed into buffer H (50 mM Tris/HCl, pH 8, 20 mM DTT) containing 50 mM NaCl. The column was eluted at 1 ml/min with a 60-ml gradient of 50 to 400 mM NaCl in buffer H. The pure protein fractions were pooled and concentrated to a protein concentration of 5 mg/ml. The His and T7 tags were removed by room temperature cleavage with a 1:1000 to 1:2000 ratio of enterokinase (Boehringer Mannheim) to subdomain protein (ratio determined in pilot runs) in the presence of 0.25 mM PMSF. After about 3 h, proteolysis was stopped by addition of 10 vol of buffer I (50 mM K-PO 4 buffer, pH 7.4, 25 mM KCl, 1.25 M (NH 4) 2SO 4, and 20 mM DTT) and the material loaded onto a 5-ml phenyl Sepharose HP (Pharmacia) column equilibrated in this buffer. After washing, the column was eluted with a 75-ml gradient to 0 M (NH 4) 2SO 4. The protein-containing fractions were pooled, concentrated to 1–2 mg/ml, and dialyzed into 50 mM K-PO 4 buffer, pH 7.5, 50 mM KCl, 10% glycerol, and 20 mM DTT. The final sample was frozen and stored at 270°C. Enzyme Assays and Kinetic Analysis IMPDH activity assays used to follow the purification of wild-type and core domain IMPDH were performed at room temperature as described (6). Formation of XMP (e 5 5400 M 21 z cm 21) was followed at 290 nm in crude fractions and formation of NAD (e 5 6220 M 21 z cm 21) was followed at 340 nm in purified fractions. For the kinetic analysis assays were performed at 37°C in 200 ml of 100 mM Tris HCl, 10 mM KCl, 3 mM EDTA, 2 mM DTT, pH 8.0, buffer. The reaction was started by addition of enzyme (15–30 nM) and the rates were obtained by monitoring NADH formation at 340 nm for 15 min in a 96-well microtiter plate reader. Michaelis constants for IMP and NAD were calculated from substrate titration data at saturating concentration of the other substrate using nonlinear least squares fitting to the Michaelis–Menten equation. For the determination of inhibition constants, saturating concentrations of both substrates were used (400 mM IMP, 400 mM NAD) and the data were fitted to the equation for uncompetitive tight-binding inhibition using the program KineTic 3.0 (8). MPA was obtained from Calbiochem. Gel Filtration To determine the degree of oligomerization, samples (150 mg in 200 ml) of wild-type, core domain, and sub-

BIOCHEMICAL ANALYSIS OF IMPDH

domain human IMPDH (II) as well as standard proteins were fractionated at a flow rate of 0.5 ml/min on a Superdex 75 HR 10/30 column (Pharmacia) equilibrated in 50 mM Tris/HCl, pH 8, 100 mM KCl, 10% glycerol, 20 mM DTT. CD Spectropolarimetry Human IMPDH (II) core domain and subdomain proteins were dialyzed into CD buffer (100 mM potassium phosphate buffer, pH 8, 10% glycerol, 2 mM b-mercaptoethanol). To adjust the protein concentration of the dialyzed samples to about 2 mg/ml, the core domain protein was diluted with dialysis buffer, whereas the subdomain protein was concentrated after dialysis in a Microcon-3 concentrator (Millipore) and centrifuged for 5 min at 16,000g in an Eppendorf centrifuge. Protein concentrations were determined by UV spectroscopy using specific molar extinction coefficients at 278 nm of 23,800, 21,000, and 2,800 M 21 z cm 21 for wild-type, core domain, and subdomain proteins, respectively (determined according to (9)). The CD data were acquired on a Jasco J-715 spectropolarimeter as described (10). Twenty scans were averaged for each protein sample and the buffer control (100 nm/min scan rate, response time 2 s) and the scans were smoothed by fast Fourier transform methods using Jasco system software. After subtracting the buffer control the data were converted to mean-residue molar elipticities using the following molecular weight and number of amino acids, respectively: wild-type, 55,804, 513, core domain protein, 40,769, 380; subdomain protein, 16,727, 147. To predict the secondary structure content of the different proteins the spectra were analyzed using the variable fitting method of Johnson and co-workers (11,12), implemented in a computer program. Miscellaneous Polyacrylamide gels were purchased from Novex and run with SDS-containing buffer according to Laemmli et al. (13). Protein determinations during purifications were performed according to the published procedure of Bradford et al. (14) using reagent from Bio-Rad and BSA as a standard. The concentrations of purified proteins were determined by UV spectroscopy as described under CD Spectropolarimetry above. The N-termini of full-length and core domain proteins were identified by Edman degradation as lacking the initiator Met residue. To estimate the amount of subdomain fusion protein in the different fractions during purification, samples were run on an SDS gel and blotted onto nitrocellulose, and the blots incubated with antibody directed against the His tag (mouse monoclonal from Clontech) and a secondary antibody coupled with alkaline phosphatase (goat anti-mouse–alkaline phosphatase conjugate, Promega) and stained. Purified sam-

ples from the MonoQ pool of known concentration served as the standard.

285 protein

RESULTS

A schematic representation of the three different transcripts used in our studies is shown in Fig. 1. The purification of human IMPDH (II) is summarized in Table 1. Gel filtration on Sephacryl S-300 was used as the last step in the purification of wild-type protein to remove enzymatically active protein aggregates. The construct encoding the core domain of human IMPDH (II) was designed by selecting the residues which form the border to the subdomain from the X-ray crystal structure of hamster IMPDH (II). The relative positions of these two residues to each other were used as a template to search the Brookhaven Protein Data Bank in order to find a suitable linker. The linker Ile-Arg-Thr-Gly was used. The protein was overexpressed in E. coli and purified as described under Materials and Methods. The purification is summarized in Table 1. In preliminary experiments it was found that the core domain protein aggregates quite readily at the salt concentration of 300 mM KCl used during the purification of the wild-type enzyme. Therefore, the KCl concentration was reduced to 100 mM KCl. It was also found that after affinity purification the protein would aggregate in the absence of IMP or on ice (not shown). The temperature-dependent aggregation appeared to be reversible. For these reasons after affinity purification the core domain protein was concentrated without further purification at room temperature and quickly frozen at 270°C until use. As indicated in Table 1, during purification, the IMPDH activity of the core domain protein could be readily determined and a more detailed characterization of the kinetic parameters of the core domain in comparison to wild-type enzyme was performed. The Michaelis and first-order rate constants were

FIG. 1. Schematic representation of IMPDH (II) transcripts described in this work: full-length human IMPDH, hIMPDH core domain, and hIMPDH subdomain.

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TABLE 1 Purification of Proteins Used in This Study

Protein

Volume (ml)

Step

Recombinant human IMPDH (II)

Recombinant human IMPDH (II) core domain

Protein (mg)

Total units (mmol/min)

Specific activity (mmol/min a mg)

Yield (%)

Purification (fold)

Soluble extract (NH 4) 2SO 4 IMP affinity pool S300

160 200 245 180

3067 1360 93 70

43.6 57.8 22.3 11.4

0.014 0.042 0.24 0.163

— 133 38.6 51.1

— 3 17.1 11.6

Soluble extract (NH 4) 2SO 4 IMP affinity pool

120 47.5 36

1512 442 108

20.16 17.91 17.93

0.013 0.04 0.166

— 88.8 100

— 3.2 12.8

Subdomain (mg) Recombinant human IMPDH (II) subdomain

Homogenate Soluble extract Talon pool MonoQ pool

190 180 27 25

2300 2200 55 16.8

18

2.8

39 a 18 a 19.3 a 16.8

— 46 107 87

— 22 21 59

2.8

—

—

Proteolysis Phenyl Sepharose a

Estimated from Western blot.

calculated from titration data obtained by monitoring the rate of NADH production at 340 nm at 37°C. The kinetic parameters of wild-type IMPDH and the core domain protein—summarized in Table 2—are very similar to each other. In addition, the tight-binding uncompetitive inhibitor MPA has very similar inhibition constants for both enzymes. These results suggest that the subdomain does not influence the overall fold and enzymatic activity of the core domain. The results are in good agreement with the kinetic parameters for human IMPDH (II) previously reported (15). The subdomain of human IMPDH (II) was expressed as a T7- and His-tagged fusion protein to facilitate purification. The subdomain fusion protein was puri-

TABLE 2 Kinetic Analysis of Human IMPDH (II) Full-Length and Core Domain Proteins

Protein

K m IMP (mM)

K m NAD (mM)

kcat (s 21)

k ii MPA (nM)

Full length Core domain

16 6 5.7 a 14 6 5.3

23 6 5.7 35 6 6

0.61 6 0.06 0.8 6 0.08

9.4 6 1.5 11.3 6 4

a Each value is an average 6 SD of at least three independent determinations.

fied from the 43,000g supernatant of E. coli lysate by metal-chelate and ion-exchange chromatography. Purification of the subdomain fusion protein is summarized in Table 1. The purified fusion protein was cleaved with enterokinase to remove the T7 and His tags and the proteolysis stopped by addition of K 1- and (NH 4) 2SO 4 -containing buffer as described under Materials and Methods. Proteolyzed subdomain was purified through hydrophobic interaction chromatography on phenyl Sepharose HP. N-terminal peptide sequencing of the proteolyzed subdomain revealed that enterokinase cleavage did not occur at the authentic site but rather 2 amino acids toward the N-terminus at an Arg residue. After cleavage, the subdomain protein contained nonauthentic Ser and Ala residues at the N-terminus. Figure 2 shows the different fractions of the purification of the subdomain fusion protein and the subdomain. Lane 5 is wider than the other lanes because the sample contains a high salt concentration (to stop enterokinase proteolysis, see above). Figure 3 shows samples of human IMPDH (II) full-length, core domain, and subdomain proteins run on a SDS gel. To determine the quaternary structure of the modules of IMPDH in comparison to full-length enzyme, gel filtration on a Superdex 75 HR 10/30 column was

BIOCHEMICAL ANALYSIS OF IMPDH

FIG. 2. Fractions from the different stages of the purification of human IMPDH (II) subdomain protein were run on a SDS gel. Lane 1, 12 mg homogenate; lane 2, 14 mg soluble extract; lane 3, 6 mg Talon pool; lane 4, 3 mg MonoQ pool; lane 5, 7.5 mg enterokinase-cleaved subdomain; lane 6, 1.9 mg purified cleaved subdomain. Molecular weight markers (kDa) are shown in lane M.

performed (not shown). The gel filtration column was calibrated with marker proteins. Both full-length and core domain proteins migrated very similarly and near the exclusion volume of the gel filtration column. It has been well established that wild-type IMPDH forms a tetramer, although higher order aggregates are also formed (for discussion see (16)) and our results suggest that the core domain protein also forms a tetramer. Based on the elution volume of proteins of known molecular weight, the molecular weight of the subdomain was calculated from its elution volume to be 24 kDa, between the expected molecular weight of a monomer of the cleaved subdomain protein of 16,727 Da and a dimer of the protein. The far UV CD spectra of full-length, core domain, and subdomain proteins were recorded. As shown in

FIG. 3. Two micrograms each of human IMPDH (II) and core domain and 1.3 mg of subdomain protein as well as markers (with molecular weight in kDa shown) were run in lanes 1–3 and M of an SDS gel, respectively.

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Fig. 4 the spectra of full-length human IMPDH and the core domain protein are very similar to each other. The spectra have a prominent negative signal at 220 nm and a positive signal at 195 nm. This result suggests that the content of secondary structure is comparable between the full-length and core domain proteins of IMPDH. In contrast, the spectrum of the subdomain protein has only one (negative) signature at 203 nm, indicating that the secondary structure of the subdomain is different than that of the core domain. These conclusions are supported by the results of the secondary structure analyses derived from the spectra (shown in Table 3). The subdomain protein contains less a-helix and b-sheet secondary structure and more turns and other types of secondary structure than both fulllength and core domain proteins, which are very similar to each other in secondary structure content. The results presented in Table 3 for full-length and core domain proteins are very similar to those reported for the secondary structure of hamster wild-type IMPDH (II) (30% a-helix, 25% b-sheet, 18% turns) (9), which in turn are in good agreement with the secondary structural elements determined in the X-ray crystal structure of the protein (4). DISCUSSION

We have described the cloning, expression, and purification of full-length human IMPDH (II) as well as the a/b-barrel core domain and the subdomain. The two-domain structure of IMPDH had been shown in the high-resolution X-ray crystal structure of the fulllength enzyme solved recently at Vertex (4).

FIG. 4. CD spectra of human IMPDH (II) (——), core domain (. . . ) and subdomain (- - -) proteins recorded as described under Materials and Methods.

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TABLE 3 Secondary Structural Elements of Human IMPDH (II) Full-Length, Core Domain, and Subdomain Proteins Deduced from Fitting of CD Spectra a-Helix

Anti-parallel b-sheet

Parallel b-sheet

b-Turns

Other

Total

Full length

33 6 1

20 6 3

961

16 6 1

22 6 2

100

Core domain

30 6 1

23 6 2

760

15 6 1

25 6 1

100

Subdomain

15 6 1

11 6 3

461

36 6 1

33 6 1

100

Protein

29 30 15

The core domain of human IMPDH (II) shares many of the biochemical properties of the full-length protein. Our results from gel filtration analysis show that the core domain forms a tetramer, as would be expected from the crystal structure. The CD analysis confirms that the core domain is structurally intact since the overall content of the secondary structure of this module of the IMPDH enzyme is very similar to that of the full-length protein (Fig. 3 and Table 3). The enzymatic parameters of this core domain are also very similar to those of full-length human IMPDH (Table 2). These findings are in accord with published results on a naturally occurring IMPDH from Borrelia burgdorferi, which does not contain a subdomain (16) and yet maintains enzymatic activity. However, this is the first study to formally show that the subdomain is not required for enzymatic activity of human IMPDH (II). Given the biochemical and enzymological data presented it is not surprising that we were unable to show a physical interaction between the core domain and subdomain proteins as analyzed by comigration on the Superdex 75 column (not shown). It has been reported that the subdomain of IMPDH is a member of the so-called cystathione-b-synthetase (CBS) protein domain family (17). The CBS domain consists of 60 residues and was originally described in the genome sequence of Methanococcus janaschii. The subdomain of IMPDH consists of two copies of the CBS protein domain arranged head to tail. A recent X-ray structure of IMPDH from S. pyogenes shows the structure of the CBS dimer domain. Each of the two CBS domains is formed by two a-helices and four strands of b-sheet structure. The function of CBS domains in general and of the subdomain of IMPDH in particular is unclear at this time. Based on the fact that mutations in the CBS domain of cystathione-b-synthase lead to human disease, a regulatory function for this protein domain has been proposed (17). The subdomain may function in the regulation of IMPDH enzyme activity. In fact, based on the alignment of 56 IMPDH sequences, only IMPDH

from B. burgdorferi lacks the subdomain (16 and data not shown), suggesting that it does play some functional role. It is also possible that the subdomain forms a module through which IMPDH interacts with other proteins, for example, GMP synthase, the next enzyme in the biochemical pathway. Other enzymatic activities in the purine biosynthesis pathway— glycinamide ribonucleotide synthase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthase—are contained in one polypeptide chain in eukaryotes (19). A physical interaction between IMPDH and other enzymes of the same pathway would fit into that frame work. It may be possible to use the subdomain or the subdomain fusion protein described in this study to investigate potential interactions with other proteins. We have observed that the core domain protein reversibly aggregates in the cold and is rather unstable in the absence of IMP. Similar properties were not observed with full-length human or hamster (II) IMPDH or with several point mutants of IMPDH that had been created for enzymological characterization of the enzyme (4). A third possible function of the subdomain then may be to improve the biophysical properties of the core domain— containing all the enzymatic machinery necessary for IMPDH function—through an increased number of exposed charged residues. Whereas the full-length enzyme contains about 25% charged residues (Asp, Glu, His, Lys, Arg), the subdomain of human IMPDH contains 37% charged residues. This speculation is consistent with the fact that the subdomain of IMPDH proteins from different species varies considerably in size and that the subdomain sequences are much less conserved than the core domain sequences. It can not be excluded, however, that the core domain described here tends to aggregate due to the specific design of the expression construct. In conclusion, we have successfully dissected the architecture of human IMPDH (II) and obtained soluble and—in the case of the core domain— enzymati-

BIOCHEMICAL ANALYSIS OF IMPDH

cally fully active modules. Given the disorder mainly in the subdomain of the crystal structure of hamster IMPDH (II) (4), the core domain protein may lead to an improved X-ray structure of the catalytically active part of IMPDH. Both domains will be valuable tools in more precisely defining the structure and function of this enzyme, which is an important target for immunosuppressive and antiviral drugs. REFERENCES 1. Collart, F. R., and Huberman, E. (1988) Cloning and sequence analysis of the human and Chinese hamster inosine-59-monophosphate dehydrogenase cDNAs. J. Biol. Chem. 263, 15769 – 15772. 2. Wu, J. C. (1994) Myophenolate mofetil: Molecular mechanism of action. Perspect. Drug Discov. Des. 2, 185–204. 3. Streeter, D. G., Witkowski, J. T., Khare, G.P., Sidwell, R. W., Bauer, R. J., Robins, R. K., and Simon, L. N. (1973) Mechanism of action of 1- b -D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole), a new broad-spectrum antiviral agent. Proc. Natl. Acad. Sci. USA 70, 1174 –1178. 4. Sintchak, M. D., Fleming, M. A., Futer, O., Raybuck, S. A., Chambers, S. P., Caron, P. R., Murcko, M. A., and Wilson, K. P. (1996) Structure and mechanism of inosine monophosphate dehydrogenase in complex with the immunosuppressant mycophenolic acid. Cell 85, 921–930. 5. Nijkamp, H. J., and De Haan, P. G. (1967) Genetic and biochemical studies of the guanosine 59-monophosphate pathway in Escherichia coli. Biochim. Biophys. Acta 145, 31– 40. 6. Fleming, M. A., Chambers, S. P., Connelly, P. R., Nimmesgern, E., Fox, T., Bruzzese, F. J., Hoe, S. T., Fulghum, J. R., Livingston, D. J., Stuver, C. M., Sintchak, M. D., Wilson, K. P., and Thomson, J. A. (1996) Inhibition of IMPDH by mycophenolic acid: dissection of forward and reverse pathways using capillary electrophoresis. Biochemistry 35, 6990 – 6997. 7. Chambers, S. P, Prior, S. E., Barstow, D. A., and Minton, N. P. (1988) The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68, 139 –149. 8. Morrison, J. F. (1969) Kinetics of the reversible inhibition of

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