1Dipartimento di Scienze Veterinarie, 3Dipartimento di Biologia MCA, 4Dipartimento di Scienze Chimiche, 7Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Università di Camerino, 5Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata and INFM, Italy and 6Institute of Molecular Biology, University of Copenhagen, Denmark
2 To whom correspondence should be addressed. e-mail: silvia.vincenzetti{at}unicam.it
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Abstract |
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Keywords: cytidine deaminase/modelling/subunit dissociation
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Introduction |
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The molecular structure of the dimeric CDA from Escherichia coli has been established (Betts et al., 1994) as has the crystal structure of the tetrameric CDA from Bacillus subtilis (Johansson et al., 2002
). The overall structure as well as the mechanism of catalysis of the dimeric and the tetrameric enzyme are very similar. The dimeric CDA from E.coli (Betts et al., 1994
) contains one active site per 31.5 kDa subunit and is composed of a small N-terminal alpha-helical domain, with no connections to the active sites, a catalytic domain containing the active site with a zinc atom co-ordinated by two cysteine residues and one histidine residue, and a C-terminal domain containing a cavity described as a broken active site. The two larger core domains have nearly identical tertiary structures and are related by approximate two-fold symmetry, but lack amino acid sequence homology. Furthermore, the overall structure of the dimer shows that the active site in one subunit is completed by contribution from the broken active site of the other subunit. The homotetrameric CDA from B.subtilis (Johansson et al., 2002
) contains four active sites; the monomer 14.9 kDa presents significant similarities with the catalytic domain of the E.coli enzyme. The zinc ion in the active site is co-ordinated with three negatively charged cysteine residues. Furthermore, residues from three subunits contribute to each active site through complicated intersubunit interactions.
Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binding site on each subunit is independent (Vincenzetti et al., 1996). Further studies by site-directed mutagenesis and chemical modification (Cambi et al., 1998
) of amino acid residues involved in catalysis have indicated that the zinc atom is co-ordinated by three cysteines (C65, C99, C102) and that a glutamic acid residue in position 67 plays an important role in catalysis. Recently, it has been shown by site-directed mutagenesis and fluorescence studies (Vincenzetti et al., 2000
) that the residues F137 and F36 may be important in stabilizing hydrophobic interactions between the ligand and the enzyme, and thereby facilitate the catalytic process. The dissociation of an oligomeric protein into monomers usually leads to changes in their functional properties (Perutz, 1989
). Therefore, identification of the residues involved in the intersubunit contacts and their eventual role in the assembly of the active site(s) is fundamental to understand the structurefunction relationship of an enzyme. In this work, we have studied the dissociation and re-association of the four subunits of human CDA. The dissociation process induced by small amount of SDS was studied by following changes in the catalytic activity and in the hydrodynamic properties of the enzyme. Fluorescence and circular dichroism (CD) spectroscopy were also used to reveal conformational changes involving the protein secondary and tertiary structure. With B.subtilis CDA (Johansson et al., 2002
), it was demonstrated that residue F125 (homologous to F137 of the human CDA) is quite close to the subunit interface of each dimer forming the tetrameric structure. Since amino acid replacement at the subunit interface may affect the enzyme function, we investigated the effect of the mutation F137W on the assembling of the subunits into a catalytically active homotetramer. Considering that the phenylalanine fluorescence is not sensitive to change in the surrounding environment and that the human CDA W113 residue is the only tryptophanyl present in each monomer, we used the double mutant, F137W/W113F to study the subunit dissociation process in the human enzyme.
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Materials and methods |
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Cytidine (CR), deoxycytidine (CdR), Tris (hydroxymethyl) aminomethane (Trizma base), dithiothreitol (DTT) and isopropyl 1-thio-ß-D-galactopyranoside (IPTG) were from Sigma Chemical Co. (St Louis, MO). Other chemicals were reagent grade from J.T. Baker Chemicals B.V. (Deventer, The Netherlands). PM10 membranes were from Amicon Corporation; Superdex 75 HR10/30 was from Pharmacia Biotech (Uppsala, Sweden); Affigel 102 and protein markers were obtained from Bio-Rad Laboratories (Hercules, CA).
Enzymes preparation
Escherichia coli SØ5201 (MC 1061cdd::Tn10 pyrD::Kan), containing plasmids pTrcHUMCDA (Vincenzetti et al., 1996), pTrcHUMCDAW113F or pTrcHUMCDAF137W/W113F (Vincenzetti et al., 2000
) were used as sources of wild-type and mutant CDAs. Cells were grown exponentially at 37°C in L-broth supplemented with 100 µg/ml of ampicillin and CDA expression was induced during late exponential growth by the addition of 1 mM IPTG. After 19 h with vigorous shaking, cells were harvested by centrifugation, washed with 0.9% NaCl, resuspended in buffer A (50 mM TrisHCl pH 7.5; 1 mM DTT, 1 mM EDTA) and lysed by sonic disruption. After centrifugation at 10 000 g for 10 min, the supernatant was heat treated at 70°C for 10 min, rapidly cooled and centrifuged again at 27 000 g for 20 min. The supernatant was applied on an affinity column containing the cytidine analogous CV6 (Cacciamani et al., 1991
) bound to an agarose resin, Affigel 102, equilibrated with buffer A. The column was washed and eluted as described by Cacciamani et al. (Cacciamani et al., 1991
). The final preparation of wild-type CDA and F137W/W113F mutant enzyme was judged >98% homogeneous by SDSPAGE. The purified enzyme, after dialysis against buffer A, was used in all experiments.
Other analytical procedures
The zinc content was determined by inductively coupled plasma optical-emission spectrometry (ICP-OES) using a Jobin Yvon 24R instrument. All measurements were performed under metal-free conditions (Vincenzetti et al., 1996). Protein concentration was determined by the Bradford protein assay (Bradford, 1976
).
Enzyme assay and stability in presence of SDS
CDA activity was assayed spectrophotometrically as previously described (Cacciamani et al., 1991). The stability of the enzyme in the presence of SDS was determined by incubating the enzyme in presence of the detergent in a final volume of 20 µl in with different SDS:enzyme molar ratios. After 2 h of incubation at 25°C, aliquots were taken and assayed for CDA activity. Renaturation of the enzymes after SDS treatment was initiated by 100-fold dilution of the denatured samples into buffer A or dialysing the mixture by ultrafiltration against buffer A.
Polyacrylamide gel electrophoresis experiments
Polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli (Laemmli, 1970), using 15% acrylamide and the Mini Protean II apparatus (Bio-Rad, gel size 7 x 8 cm x 0.75 mm). The markers used were Bio-Rad low range. To study the dissociation of CDA into subunits, a series of 15% PAGEs were done in the presence of increasing concentrations of SDS, ranging from 0.35 to 1.73 mM both in the gels and in the running buffer. The samples were prepared as follows: 3.6 µg of each protein sample were incubated in presence of SDS ranging from 0.35 to 1.73 mM in order to reach different molar SDS:enzyme ratios. After 2 h of incubation at 25°C, Bromophenol Blue was added and the sample was loaded on a gel. The electrophoresis was performed at 4°C with a constant voltage of 200 V.
The effect of the strong CDA inhibitor F-ZEB on dissociation was studied as follows: 4.0 µg of enzyme were incubated with F-ZEB at final concentrations ranging from 0.06 to 60 µM. After 3 h at 25°C, SDS was added to a final concentration of 1.73 mM (SDS:enzyme molar ratio = 200). The mixture was incubated for an additional 2 h at 25°C and subsequently subjected to 15% PAGE in the presence of 1.73 mM SDS. W113F mutant enzyme was used as control in all experiments. CDA was visualized on the gel by Coomassie Blue. In certain experiments, the gels were cut into 2 mm slices and each slice extracted for 2 h in buffer A containing the same percentage of SDS as used during electrophoresis. CDA activity in the eluate of each slice was determined as described above.
To measure the relative amount of the tetramer at each SDS:enzyme ratio, both in the wild-type CDA and mutant enzyme, semi-quantitative analyses on the electrophoretic gels were performed. The separating gels (prepared as described above) were scanned and analysed with QUANTISCAN software (BIOSOFT, Ferguson, MO). A graphic presentation of protein zones as peaks was produced and from the calculated peak areas the percentage of the tetrameric and monomeric form was calculated.
Reactivation of CDA
Monomeric CDA eluted from a 15% PAGE gel containing 1.73 mM SDS, as described in the previous section, was dialysed against buffer A under metal-free conditions (Vincenzetti et al., 1996), analysed for enzymatic activity and zinc content, and run again on 15% PAGE, 0.35 mM SDS to determine the extent of re-association.
Spectroscopic measurements
The CD experiments were performed with a Jasco J710 spectropolarimeter at 25°C in the far (90250) and in the near (250320) UV range. Buffer was 0.02 M TrisHCl pH 7.6, 1 mM DTT; protein concentration was 2 µM in the far UV range (cell length 2 mm) and 20 µM in the near UV range (cell length 10 mm). Molar ellipticity [] (deg cm2 dmol1) is expressed on a mean residue concentration basis in the far UV region and on a protein concentration basis in the near UV region. All spectra were corrected by baseline subtraction.
Fluorescence measurements were performed on a Fluoromax 3 (Jobin Yvon) spectrofluorometer, equipped with an external bath circulator at 25°C. The steady-state emission spectra were corrected by blank subtraction in order to eliminate Raman scattering effects. The buffer used in the experiments was 20 mM TrisHCl pH 7.6 containing 1 mM DTT. The excitation wavelength was 280 nm.
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Results |
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A time course of the inactivation of CDA by low concentration of SDS (ranging from 0.35 to 1.73 mM) was performed. The results obtained indicated that the process reached equilibrium after 2 h at 25°C (data not shown). At different SDS:enzyme molar ratios, the inactivation of both wild-type and the F137W/W113F mutant enzyme was non-cooperative as demonstrated in Figure 1a. The same experiment conducted on the W113F mutant enzyme showed an inactivation curve similar to that of the wild-type CDA (data not shown). Both wild-type CDA and the W113F mutant enzyme reached complete inactivation at an SDS:enzyme molar ratio of 800, whereas the F137W/W113F mutant enzyme seemed more sensitive and was completely inactivated at an SDS:enzyme molar ratio of 250. CDA activity was immediately recovered following a 100-fold dilution of the SDS-treated samples into renaturation buffer (see Materials and methods).
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The dissociation of human CDA into subunits was studied by 15% PAGE performed at different SDS concentrations. As shown in Figure 1b, the wild-type CDA was a tetramer with a relative migration (Rm) of 0.19 up to an SDS:enzyme molar ratio of 61; at an SDS:enzyme ratio of 123, it started to dissociate into monomers with an Rm of 0.72 (lane 2). The dissociation was completed at an SDS:enzyme ratio of 616 (lane 4). Intermediate forms such as trimers and/or dimers were not observed. With the F137W/W113F mutant enzyme, the dissociation process was significantly faster. Figure 1b shows that at an SDS:enzyme ratio of 31, the enzyme was present only in the tetrameric form (lane 5), whereas it existed predominantly as monomer at an SDS:enzyme ratio of 62 (lane 6). The dissociation process in the mutant enzyme was completed at an SDS:enzyme ratio of 154 (lane 8). Measurements of the CDA activity eluted from 2 mm gel slices following electrophoresis showed that only the tetrameric form of CDA retained activity, while the monomer was completely inactive (data not shown). The inactive monomeric enzyme extracted from the gel was reactivated after extensive dialysis, under metal-free conditions, against buffer without the dissociating agent. ICP analysis on the reactivated CDA revealed the presence of 1 mol zinc per mol of enzyme subunits. Furthermore, the reactivated enzyme showed one single band, corresponding to the tetrameric form of CDA upon PAGE run under native conditions (data not shown). The mutant enzyme W113F was used as control in all the experiments and its dissociation pattern resemble that of the wild-type CDA (data not shown).
Semi-quantitative analysis of the PAGE images allowed the calculation of the SDS:enzyme ratio that caused 50% dissociation of the tetrameric form of the enzyme into monomer. This value was 316 for the wild-type CDA and 69 for the mutant enzyme (data not shown). The presence of the strong CDA competitive inhibitor 5-fluorozebularine (F-ZEB, 5-fluoropyrimidine-2-one ribonucleoside) affected the subunit dissociation of wild-type CDA (Figure 2a, lanes 14); in fact, at an SDS:enzyme molar ratio of 200, the wild-type CDA is predominantly present in the monomeric form, but addition of increasing amounts of F-ZEB ranging from 0.06 to 60 µM resulted in the disappearance of the monomer and increased amounts of the tetrameric form. On the contrary, the presence of the inhibitor did not affect the F137W/W113F mutant enzyme subunit dissociation (Figure 2a, lane 5). By scanning the gels, the percentage of the monomer at each F-ZEB concentration was calculated and from the graphic representation of the data (Figure 2b), it was deduced that 1.2 µM of the inhibitor was required to reduce the dissociation of the tetramer to 50%, under the conditions employed.
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The CD spectra in the far UV region (190250 nm) of wild-type and mutant F137W/W113F CDA revealed great similarities in the secondary structure organization of the two proteins (Figure 3), indicating that the double mutation does not affect the overall structure of the protein. In contrast, CD spectra in the near UV region showed clear differences between the two proteins (Figure 3, inset). The spectrum of the mutant protein completely lacked optical activity between 250 and 290 nm. As the CD signal in this region arises from asymmetric interactions of aromatic amino acids with nearby groups (Strickland, 1974) provided by the protein matrix, this result and the peak position in wild-type CDA (267 nm) suggest that the substitution of F137 with a larger amino acid side chain (such as a tryptophan) induced a small but detectable perturbation in the local tertiary structure. The presence of increasing concentration of SDS caused a slight decrease in the secondary structure content for both wild-type and mutant CDAs (data not shown). This effect may be ascribed to the SDS-induced dissociation revealed by PAGE and the gel filtration experiments performed under the same conditions. A similar effect has been observed with other proteins (Kelly and Price, 1987
; Gahn and Roskoski, 1995
). In Figure 4, the ratio between the ellipticity at 222 and 208 is reported at different SDS concentrations. As shown, the monomerization process differed for the two homotetrameric proteins: the quaternary structural organization of the mutant protein seemed more affected as increasing SDS concentration leads to a steeper decrease in secondary structure content than for the wild type; around an SDS:enzyme molar ratio of 500, the secondary structure content became stable. Surprisingly, when we overcame the critical micellar concentration of SDS (cmc = 2.2 mM) (Takeda et al., 1985
), CD spectra showed a significant recovery of secondary structure probably due to the formation of a compact micelle that protected the exposed hydrophobic surfaces of each monomer by mimicking the quaternary structure interactions: as a control, no activity was evident for these samples. Tetramer to monomer dissociation of both wild-type and double mutant CDA was also followed by fluorescence spectroscopy. The steady-state fluorescence spectrum of wild-type CDA (Figure 5) peaked at
335 nm, which is a typical emission wavelength for a partially buried tryptophan residue (Burstein et al., 1973
). The double mutant spectrum was shifted to longer wavelengths (
348 nm), indicating that the polarity around the tryptophan residue at position 137 is much higher than that at position 113, i.e. its original position in the wild-type protein. Interestingly, the wild-type CDA spectrum exhibits a shoulder at
309 nm, which is totally absent in the mutated protein. Since the measurements depicted in Figure 5 were done with an excitation wavelength of 280 nm, this contribution in the blue-edge of the fluorescence spectrum may be ascribed to tyrosine fluorescence, which is typically peaked at
303305 nm (Lakowicz, 2000
). This conclusion is also supported by the disappearance of the 309 nm shoulder when the sample was excited at 295 nm (data not shown), where tyrosine emission is negligible. The addition of SDS yielded different results for the two samples. In the case of wild-type CDA, a slight broadening of the spectrum was observed, without any major change in the total fluorescence intensity. On the other hand, a much larger effect was obtained with the double mutant, including a dramatic decrease of the signal at 349 nm upon SDS-induced monomerization, as well as the appearance of a pronounced shoulder at 305 nm.
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Discussion |
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Site-directed mutagenesis is in progress to modify the highly conserved residues Y60 (corresponding to the E.coli Y633 and B.subtilis Y48) and Y33 in order to better clarify how this residue may participate in the intersubunit interactions together with the F137 residue.
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Acknowledgements |
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References |
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Received February 10, 2003; revised October 14, 2003;; accepted October 21, 2003