From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
Received for publication, March 21, 2003 , and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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The intermediate domain sequence as a whole has no identity to any known protein. A data base search revealed, however, that the fragment between residues 366 and 405 in the intermediate domain is 35% identical to a sequence from calmodulin-like protein identified in rice (10). The identity rises to 45.5% for residues 384405 (Fig. 2A). It is interesting that in the calmodulin structure this sequence forms a long central -helix that serves as a linker connecting two globular domains of calmodulin (11). The crystal structure of calmodulin (12, 13) showed that the helix is straight and is not surrounded by any other
-helices or
strands (Fig. 2B). NMR studies of calmodulin, however, have revealed a non-helical region at residues 7881 (14). It has been further shown that the central helix is disrupted into two helices connected by a long flexible loop (residue 7482, Fig. 2C) (15). Thus, rather than a rigid spacer, the linker serves more as a flexible tether between two domains, allowing them to swing around (16, 17). Movement in the central helix changes calmodulin conformation from an elongated dumbbell to a compact globular form (18). Such conformational changes occur during binding of target peptides (15), which is an important mechanism in calmodulin function (16, 19, 20).
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Although the similarity of the intermediate domain with calmodulin (Fig. 1A) is lower than with the calmodulin-like protein (36.4% and 45.5%, correspondingly), it is 63.6% in both cases if conservative changes are taken into account. We suggest that the "calmodulin-like" region of the intermediate domain with its flexible central part plays an important role in coupling the two catalytic domains of FDH to produce dehydrogenase activity. In the present work we apply a site-directed mutagenesis approach to explore the role of this region in FDH function.
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EXPERIMENTAL PROCEDURES |
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Generation of the Construct for Intermediate Domain Expression The construct for the intermediate domain expression was generated in two steps from pRSET vector with cloned FDH cDNA (21). This construct has a unique NdeI restriction site upstream of the 5'-translated region of the FDH, which overlaps the ATG start codon, and a unique ScaI restriction site on 3'-translated region, which overlaps the TGA stop codon. In the first step, the second NdeI restriction site was introduced into the vector immediately upstream of the intermediate domain coding sequence using site-directed mutagenesis. The sequence between two NdeI sites was excised, and vector was religated through internal NdeI sites. This approach has created an in-frame ATG start codon next to which was a triplet coding for the first amino acid in the intermediate domain sequence. In the second step, the second ScaI restriction site was introduced into the vector immediately downstream of the intermediate domain coding sequence using site-directed mutagenesis. The sequence between two ScaI sites was excised, and vector was religated through internal ScaI sites. This approach has created an in-frame TGA stop codon immediately downstream of a triplet coding for the last amino acid in the intermediate domain sequence.
Site-directed MutagenesisThe QuikChange site-directed mutagenesis kit (Stratagene) was used to generate a construct for the intermediate domain expression and to introduce desirable mutations within the intermediate domain. Site-directed mutagenesis was carried out directly on expression constructs: pRSET with cloned intermediate domain cDNA or pVL 1393 with cloned FDH cDNA (22). Experiments were performed according to the manufacturer's protocol. Plasmids were isolated by using a Quantum Prep kit (Bio-Rad) and sequenced to identify clones carrying the mutation. The entire coding region of the clones carrying the mutations was sequenced to ensure the absence of random mutations.
Expression of the Intermediate Domain and Its MutantsEscherichia coli BL21(DE3) cells (Novagen) were transformed with the corresponding expression vector according to the manufacturer's protocol, and the cells were grown in 4 ml of NZCYM medium containing ampicillin (50 µg/ml) overnight at 37 °C with shaking. The 100 ml of NZCYM medium containing ampicillin were inoculated with the overnight culture and incubated at 37 °C with shaking until A600 = 0.6 (about 6 h) followed by induction with isopropyl -D-thiogalactopyranoside (1 mM final concentration). Three hours after induction, the cells were harvested by centrifugation (5000 x g, 10 min) and resuspended in 2 ml of buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 0.1% Triton X-100). The suspension was chilled on ice and sonicated (three times for 45 s). The insoluble material was precipitated by centrifugation (18,000 x g, 15 min). The pellet was dissolved in 5 ml of 6 M urea by continuous stirring for 1 h at 4 °C. The supernatant and the dissolved pellet were examined for the presence of the protein of interest by SDS-PAGE.
Purification of the Intermediate Domain and Its MutantAfter dissolving the pellet of insoluble cell fraction in 6 M urea, the sample (0.1 mg/ml protein) was dialyzed against 20 mM Tris-HCl buffer, pH 7.5, for 18 h at 4 °C. Insoluble material was removed by centrifugation (18,000 x g, 15 min), and the soluble sample was subjected to the size-exclusion chromatography procedure on a Sephadex G-100 column (2.5 x 80 cm). The column was equilibrated with 20 mM Tris-HCl buffer, pH 7.5, containing 1 mM NaN3. About 15 mg of protein was loaded onto the column in a volume of about 3 ml. Chromatography was carried out at 20 ml/h flow rate, and fractions of 4 ml were collected. The purified preparations were stable for at least 2 weeks when stored at 4 °C or for several months if stored frozen at 20 °C.
Expression of Mutants of Full-length FDHThe expression of the full-length FDH mutants has been done in insect cells using a baculovirus expression system as we previously described (21). The vector carrying mutated FDH cDNA and linearized AcNPV viral DNA was co-transfected into Sf9 cells using the BaculoGold transfection kit (BD Pharmingen, San Diego, CA) according to the manufacturer's protocol. Recombinant viral stock was amplified in Sf9 cells to produce high titer virus stock. High Five cells were seeded as a monolayer in 25-cm2 flasks (about 2 x 106 cells per flask) and grown overnight. Each flask was infected with 0.1 ml of high titer virus stock. Culture medium and cells were collected separately 24 days post-infection, and the recombinant protein production was detected by SDS-PAGE followed by Western immunoblot analysis. For large scale recombinant protein production, High five cells were seeded in 225-cm2 flasks (about 20 x 106 cells per flask), and after growing overnight each flask was infected with 1.0 ml of high titer virus stock. After 96 h cells were collected, spun down, and rinsed with 20 mM Tris-HCl buffer, pH 7.5, containing 1 mM NaN3. Cells were resuspended in the same buffer containing protease inhibitor mixture (Sigma) and were broken by freezing/thaw procedure. Insoluble material was removed by centrifugation, and the soluble fraction of the cells was used to isolate recombinant protein.
Purification of FDH MutantsMutants of the full-length FDH having an altered intermediate domain were purified from the soluble cell fraction by size-exclusion chromatography on Sephacryl S-300 column (1.5 x 100 cm). The column was equilibrated with 20 mM Tris-HCl buffer, pH 7.5, containing 10 mM 2-mercaptoethanol, and 1 mM NaN3. About 5 mg of protein was loaded onto the column in a volume of about 3 ml. Chromatography was carried out at 20 ml/h flow rate, and fractions of 4 ml were collected.
Analysis of the Mutant ProteinsSDS-PAGE was carried out according to the method of Laemmli (23) in 8.0% or 15.0% gel. Protein concentration was determined using Bradford's protein assay (24). Amino-terminal sequence analysis of wild type and mutant intermediate domain was carried out in the Protein Core facility at the Medical University of South Carolina using automated Edman degradation with gas phase analysis on an LF-3000 (Beckman) or an Applied Biosystem 470A (PerkinElmer Life Sciences) protein sequencer.
Assay of Enzyme ActivityAll assays were performed at 30 °C in a Shimadzu 2401PC double-beam spectrophotometer. For measurement of hydrolase activity the reaction mixture contained 0.05 M Tris-HCl, pH 7.8, 100 mM 2-mercaptoethanol and 5 µM of substrate, 10-formyl-DDF. 10-Formyl-DDF is an alternative, stable substrate for the enzyme (25). The reaction was started by the addition of enzyme (120 µg) in a final volume of 1.0 ml and read against a blank cuvette containing all components except enzyme. Appearance of product 5,8-dideazafolate was measured at 295 nm using a molar extinction coefficient of 18.9 x 103 (26). Addition of NADP+ to the reaction mixture provided a measure of both dehydrogenase and hydrolase activities, i.e. total activity of the enzyme. Hydrolase activity measured in the absence of NADP+ was subtracted from the total activity to give the dehydrogenase activity. Dehydrogenase activity was also measured independently using the increase in absorbance at 340 nm due to production of NADPH and the molar extinction coefficient of 6.2 x 103. Aldehyde dehydrogenase activity was assayed using propanal essentially as described by Lindahl and Evces (27) with our modifications (2). The reaction mixture contained 50 mM CHES buffer, pH 9.4, 5 mM propanal, 1 mM NADP+, and enzyme in a total volume of 1 ml. Activity was estimated from the increase in absorbance at 340 nm.
MALDI Mass SpectrometryExpressed wild type intermediate domain and its mutants were analyzed at the Medical University of South Carolina mass spectrometry facility using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A time-of-flight MALDI (Voyager-DE STR from Applied Biosystems) was used in linear mode to detect average ion masses. Samples were prepared by mixing 1 part protein solution (0.52.5 µg/µl) with 3 parts matrix (50 mM -cyano-4-hydroxycinnamic acid in 70% acetonitrile and 0.1% trifluoroacetic acid). 0.5 µl of each sample mixture, containing 525 µmol of peptide, was then spotted and allowed to air dry prior to analysis.
Fluorescence StudiesFluorescence experiments were done on a Hitachi F-2500 fluorescence spectrophotometer. Emission fluorescence spectra of wild type intermediate domain and its mutants were recorded by scanning from 300 to 460 nm with fluorescence excitation at either 280 or 295 nm. Protein samples (0.1 mg/ml) were in 20 mM Tris-HCl buffer, pH 7.5. All experiments were done at room temperature.
Analysis of Conformation by CD SpectroscopyFar-UV CD spectra (190250 nm) were obtained using Jasco J710 spectropolarimeter (Easton, MD) at a resolution of 0.2 nm with a bandwidth of 2 nm. For each sample, ten spectra were collected and averaged. The concentration of wild type and mutant proteins was about 0.5 mg/ml in 50 mM potassium phosphate, pH 7.0, containing 10 mM NaCl. Protein concentration was determined using a calculated molar extinction coefficient (28). Spectra were recorded at 25 °C using cylindrical quartz cuvette with a path length of 0.02 cm. The protein spectra were corrected by subtracting a blank solution containing 50 mM phosphate, pH 7.0, and 10 mM NaCl. The final results are reported as mean residue ellipticity in units of degree·cm2·dmol1 obtained using the calculated mean residual mass of each derivative. The spectra were analyzed using a method allowing quantitative calculation of the content of the secondary structure elements (29).
Prediction of the Secondary StructurePredicted secondary structures of the intermediate domain and its mutants were generated by using the server of Biomolecular Engineering Research Center at Boston University (Boston, MA, available at bmerc-www.bu.edu/index.html) and the server of European Molecular Biology Laboratory (available at www.embl-heidelberg.de/predictprotein).
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RESULTS |
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Expression and Purification of Wild type and Mutant Intermediate Domains of FDHWe expressed and purified FDH intermediate domain corresponding to the original FDH sequence and intermediate domain mutants corresponding to the above mutants generated for the full-length enzyme (Fig. 2D). We found that the original intermediate domain and three other constructs were expressed as insoluble proteins. The proteins were refolded and purified from the insoluble fraction of the cell in one step by size-exclusion chromatography. Each of the four intermediate proteins was eluted from the column as single peak with apparent molecular mass of about 1014 kDa (data not shown), suggesting that they were not aggregated in solution. All constructs for the intermediate domain expression had an extra nucleotide triplet encoding for methionine in an amino-terminal end that was necessary to initiate translation. Analysis of the amino-terminal sequences of purified proteins revealed that they do not have a terminal methionine and start from serine (Ser-311 in the FDH sequence). The first methionine, however, is often not retained at the amino terminus of the proteins expressed in prokaryotes (30). Thus, the intermediate domain was expressed as an original sequence presented in the full-length FDH.
Comparison of Wild Type and Mutant Intermediate DomainsWe compared CD spectra of the intermediate domain and its mutants. These experiments were carried out to detect changes in the secondary structure elements within the domain induced by the mutations. CD spectra in the far-UV range reflect content of secondary structure components in the proteins with the strongest and most characteristic spectrum for the -helix (31). These experiments revealed significant shifts in CD plots indicating changes in secondary structure element composition (Fig. 3). The spectra were analyzed using a method that allows quantitative calculation of the content of the secondary structure elements (29). Data derived from this analysis (Table II) revealed that introduction of alanine residues in the putative flexible region resulted in an increase of
-helical content with a decrease in random coil content compared with the wild type intermediate domain. These results indicate that the entire putative helix (about 22 amino acid residues) is likely to be straight in the mutant. The other mutant, 22del, in which the putative long
-helix was deleted, showed a notable decrease in content of
-helical region compared with the wild type protein. This decrease was proportional to the relative contribution of this fragment in the
-helical portion of the intermediate domain. Results on the last mutant, 6del, suggest that the deletion of the putative flexible region within the helix stabilized the entire helix, which probably becomes straight similar to 6Ala mutant.
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To further explore whether or not conformational changes occur due to the introduced mutations, we compared the emission fluorescence spectra of the intermediate domain and its 6Ala mutant. The intermediate domain has one tryptophan and two tyrosine residues that allow one to measure its intrinsic fluorescence. We performed these experiments at excitation wavelengths of 278 and 295 nm. We observed significant differences in the fluorescence spectra of the mutant compared with the spectrum of the wild type intermediate domain (Fig. 4). There was stronger shift in the fluorescence maximum with an excitation at 278 nm (from 322 to 344 nm) than with excitation at 295 nm (from 332 to 341 nm). These results suggest that the tryptophan and the tyrosines become more susceptible to solvent in 6Ala mutant than in the wild type intermediate domain.
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Alanine-scanning MutagenesisUsing alanine-scanning mutagenesis we studied whether substitution of a single residue within the putative flexible regions will influence FDH activity or generate secondary structure changes within the intermediate domain. We have generated six mutants of the full-length FDH and six corresponding mutants of the intermediate domain protein. Thus, following mutants were expressed and purified: K394A, L395A, R396A, G397A, E398A, and D399A. The six mutants of the full-length FDH were expressed and purified as described for the wild type enzyme (22). All mutants showed a similar level of expression and the same elution profile when purified on 5-formyltetrahydrofolate affinity column (data not shown). They come off from the affinity column during elution with 20 mM folate that are the same conditions as for elution of the wild type enzyme (22). Assay of the enzyme activity of the purified mutants revealed that they all possessed three types of activities, hydrolase, dehydrogenase, and aldehyde dehydrogenase (Table I). Levels of all three activities were comparable to the activities of the wild type enzyme (Table I).
Expression of the six corresponding mutants of the intermediate domain revealed that they all were expressed as insoluble proteins similar to the wild type intermediate domain. The mutants were solubilized from inclusion bodies, refolded, and purified by size-exclusion chromatography as described above. Each of the mutants came out of the column as a single peak with an apparent molecular mass of about 15 kDa (data not shown) that is similar to the elution profile of the wild type intermediate protein. This suggests that they were not aggregated in solution. To ensure that the expressed intermediate domain was not truncated or otherwise degraded and to check for relative purity, we analyzed the wild type intermediate domain and its mutants using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Results of MALDI-MS analysis showed that all of the expressed polypeptides behaved as a single molecular mass species and matched their respective calculated molecular masses (Table III). Amino-terminal sequence analysis revealed that each of the purified intermediate domain preparations contains only the intermediate domain protein. Thus, these experiments showed that expressed intermediate proteins were not degraded from the termini. CD spectra of all six mutants of the intermediate protein were close to each other and similar to that of the wild type protein in contrast to 6Ala mutant spectra (data not shown). The calculated content of the secondary structures confirmed close structural similarity between these mutants and the wild type intermediate domain (Table IV). These results suggest that neither of these single-point mutations induce substantial changes in the secondary structure.
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DISCUSSION |
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Our experiments with site-directed mutagenesis of the intermediate domain suggest that the calmodulin long helix-like region of FDH is likely to be bent in a manner similar to that of the calmodulin structure (15, 18). All mutants of full-length FDH retained hydrolase and aldehyde dehydrogenase activities similar to those for the wild type enzyme suggesting that both functional domains themselves were not affected by the mutations. This is consistent with our previous studies that have shown that the catalytic domains are capable of folding into functional proteins independently of each other or of the intermediate domain (2, 3). However, deletion of either the entire long helix-like sequence or just the putative flexible region of the sequence, or the change of all residues in the flexible region to alanines, resulted in an enzyme that lacks dehydrogenase activity. An explanation of these results is depicted on the schematic (Fig. 5). Because alanine has the highest propensity for -helix formation (32), replacement of the residues within the flexible region with alanines has apparently straightened the helix by pulling apart the amino- and carboxyl-terminal domains. This should prevent concerted action of the two domains resulting in the loss of the dehydrogenase activity. It is less obvious what happened to the reciprocal orientation of the amino- and carboxyl-terminal domains after deletions are made within this region. We suggest that deletion of the entire "calmoduline-like" region in FDH resulted in more tight but much less flexible interactions between the two domains. Such an orientation apparently misoriented the folate binding site in the amino-terminal domain with respect to the dehydrogenase catalytic center located in the carboxyl-terminal domain (Fig. 5). We further suggest that deletion of the putative flexible region affected the reciprocal domain orientation in a manner similar to that when the region was replaced with alanine residues thus distancing the domains from each other.
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Predicted secondary structure of the intermediate domain showed indeed that regions between residues 379 and 393 and between 403 and 416 have a high tendency to form -helices but a low tendency to form either
-sheets or random coils. In contrast, the region between residues 394 and 402 has a high tendency to form a random coil. This suggests that the region is likely to be represented by two
-helices connected by a loop. For the 6Ala mutant it has been predicted that the region between residues 394 and 402 gains a tendency to form an
-helix instead of a random coil. Secondary structure algorithms predicted that this construct has a long
-helix between residues 380 and 415 that coincides with our hypothesis. We also modeled simulations of the original calmodulin-like 22-residue-long fragment and the fragment with six residue converted to alanines. This has been done using SYBYL 6.7 (TRI-POS Associates Inc., St. Louis, MO). In modeling simulations with the original fragment, adding turns to the putative six-residue flexible region results in a conformation with a lower minimum energy than with an
-helix. When the six residues were converted to alanine, the converse was true: modeled as a helix, the region reached a lower minimum energy. It should be also noted that the putative flexible region and adjacent upstream and downstream sequences are enriched with charged residues that create an opportunity for multiple salt bridge formations and hydrogen bonding. Indeed, the modeling revealed possible salt bridge formation when the putative helix is bent in the middle that apparently stabilizes it in the required conformation.
The results of CD spectroscopy studies of the intermediate domain, and its mutants expressed as separate proteins, correspond well to the predicted secondary structures. Better correspondence was observed, however, for the wild type intermediate domain and for the 6Ala mutant. Both theoretical predictions and experimental approaches showed an increase in -helical content with a decrease in random coil content in this mutant compared with the wild type protein. A significant increase in
-helix content was also observed for another mutant, 6del. This suggests that this mutation resulted in the formation of a single long helix rather than two closely located helices connected by a shorter loop. Studies on calmodulin also revealed that deletion within the flexible region results in straightening of the central helix (33). A notable decrease in the
-helix content in the 22del mutant further suggests that the region is indeed represented mainly by an
-helix. The results of fluorescence spectroscopy studies are in accordance with the results of CD-spectroscopy studies. Comparison of the fluorescence spectra of the wild type intermediate domain and 6Ala mutant revealed that the tryptophan and tyrosines of the 6Ala mutant are more exposed to the solvent than in the wild type protein suggesting that the mutant has a more open conformation. The single tryptophan (Trp-332) of the intermediate domain is distant from the sequence mutated, whereas the two tyrosines (Tyr-380 and Tyr-409) are located in close proximity to this region flanking it from each side. This explains the stronger shift in the fluorescence maximum at 278 nm versus the one at 295 nm. With excitation at 295 nm, the spectrum reflects fluorescence of tryptophans, whereas with excitation at 278 nm there is a significant contribution of tyrosine residues to the fluorescence (34). Apparently, straightening the putative helix makes the nearby tyrosines more susceptible to solvent, whereas the more remote tryptophan is less affected. Although the truncated mutants also revealed significant changes in their intrinsic fluorescence, we did not analyze them in detail. Interpretation of such results would be more difficult, because conformational changes of these mutants might be the result of a difference in size and not just the result of changes in secondary structure content. Overall, these results support our explanation of the role of the region in FDH function.
We propose that flexibility within the calmodulin long helix-like region of FDH is associated with the enzyme mechanism. Our results imply that simple connectivity between the two functional domains is insufficient to produce the dehydrogenase activity. Due to its structural organization, FDH undergoes significant conformational changes during its catalytic activity. After substrate binding, it is likely that the enzyme changes conformation and couples the two functional domains to initiate the dehydrogenase reaction. In turn, opposite changes uncouple the domains after completion of the reaction to allow release of the product. For such a mechanism, the calmodulin long helix-like region of FDH is a suitable candidate to work as a hinge, allowing reciprocal movement of the functional domains. This region might also be a site for regulation of the enzyme. Thus, stabilization of the sequence in elongated conformation will distance the functional domains "turning off " the enzyme or "switching" from the dehydrogenase to hydrolase activity.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., P. O. Box 250509, Charleston, SC 29425. Tel.: 843-792-0845; Fax: 843-792-8565; E-mail: krupenko{at}musc.edu.
1 The abbreviations used are: FDH, 10-formyltetrahydrofolate dehydrogenase; THF, tetrahydrofolate; CHES, (2-[N-cyclohexylamino]ethanesulfonic acid; 10-formyl-THF, 10-formyltetrahydrofolate; 10-formyl-DDF, 10-formyl-5,8-dideazafolate; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; CD, circular dichroism.
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ACKNOWLEDGMENTS |
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REFERENCES |
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