©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Electrophoretic and Spectral Characterization of Wild Type and Mutant Adenovirus Protease (*)

(Received for publication, April 12, 1995; and in revised form, August 11, 1995)

Hossein Keyvani-Amineh (§) Mounir Diouri J. Guy Guillemette (¶) Joseph M. Weber (**)

From the Department of Microbiology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The P137L mutation in the adenovirus type 2 protease results in a temperature-sensitive protein-trafficking phenotype expressed during infection but not in vitro. Homology-derived secondary structure prediction placed the mutation within an externally disposed loop. Circular dichroism and urea gradient gel electrophoresis suggested that, unlike other thiol proteases, the Ad2 protease is comprised of a single conformational domain. The -0.32-kcal difference in the free energy of folding and the temperature-independent CD spectra of the mutant and wild type enzymes point to a very subtle structural change as the cause of the in vivo phenotype.


INTRODUCTION

Adenoviruses, like most viruses, encode an endopeptidase that is essential for virion maturation and infectivity. The gene has been sequenced in 12 different adenoviruses and, though highly conserved, it lacks signature proteinase motifs and has no significant homology to known proteins(1, 2) . Much of our knowledge about this cysteine protease derives from studies with a temperature-sensitive Ad2 (^1)mutant, ts1, which contains a Pro to Leu mutation at residue 137 (P137L) and fails to effect the proteolytic cleavages at the non-permissive temperature (3) . We have recently shown that ts1 is not temperature sensitive in vitro and that its in vivo phenotype may be due to faulty protein trafficking at 39 °C due to the P137L mutation(4) .

In this report we examine the effect of the P137L mutation on the structure and stability of the protease by means of urea gradient gel electrophoresis and circular dichroism.


MATERIALS AND METHODS

Expression and Purification of Adenovirus Protease

Cloning, expression, and purification of the Ad2 protease using the pRSET A vector was done essentially following the manufacturer's protocols (Invitrogen, San Diego, CA). ts1 was cloned and expressed identically to wt. Purification of both proteases was carried out under denaturing conditions by metal chelating affinity chromatography (MCAC, chelating Sepharose Fast Flow from Pharmacia Biotech Inc.). Purified protease was renatured by the solid phase method(5) , the pH was adjusted to 6, and then the protease was loaded on a pre-equilibrated MCAC column. The column was washed five times with MCAC-O buffer (20 mM Tris, 0.5 M NaCl, pH 7.9), and the protease was eluted by increasing concentrations of imidazole. Protease normally eluted at 1 M imidazole. Eluted protease was dialyzed against 20 mM PO(4), 1 mM EDTA, 5 mM 2-mercaptoethanol overnight and then concentrated on Centricon 30 (Amicon, Lexington, MA) and centrifuged for 15 min at 14,000 times g to remove any precipitated protease. Protein concentration was determined by ultraviolet absorption (6) and by the method of Bradford(7) .

For circular dichroism the protease was purified by a modification of a previously described method(1) . Escherichia coli (AR120) bearing the pLPV expression vector(8) , in which the wt and ts1 proteases had been cloned, was harvested from 1 liter of medium and suspended in buffer A (10 mM Tris-HCl, pH 8.5, 10% glycerol, 10 mM beta-mercaptoethanol, 0.5 mM EDTA). This suspension was freeze-thawed for five successive cycles in methanol/dry ice slurry, followed by sonication on ice for ten 10-s bursts at a medium intensity setting. Inclusion bodies were sedimented by centrifugation at 5000 rpm for 20 min and the supernatant was kept. The sedimented inclusion bodies were dissolved in buffer A (2 volumes) saturated with urea and added to the supernatant to promote refolding. DEAE-Sephacel batch treatment was unmodified(8) .

Hydroxyapatite Column Chromatography

The DEAE-Sephacel-treated solution was dialyzed against buffer B (20 mM phosphate buffer, pH 6.7, 10% glycerol, 5 mM beta-mercaptoethanol, 0.5 mM EDTA) and loaded on a K16/20 column (Pharmacia) packed with hydroxyapatite (Bio-Rad). A constant flow rate was provided by a peristaltic pump, and the protease was eluted with a phosphate gradient.

SP-Sepharose Chromatography

Partially purified fractions of the protease were combined and dialyzed against 10 mM MES buffer containing 5 mM beta-mercaptoethanol (pH 7) and then loaded on a K16/20 column packed with SP-Sepharose. Protease was eluted with a gradient of 10 mM MES, 0.5 M NaCl buffer.

Sephacryl S-200 Gel Filtration Chromatography

As the final step of purification the protease was passed through a K16/20 column packed with Sephacryl S-200 HR (Pharmacia), and the fractions containing the protease were collected and dialyzed two times against 10 mM MES buffer. Purity was assessed by SDS-PAGE and silver staining.

Circular Dichroism

Immediately prior to CD the purified protease (pLPV vector) was centrifuged at 14,000 times g for 15 min and passed through a 0.45-µm filter. CD spectra were recorded on a Jasco 710 spectropolarimeter with a thermostated cell holder and a Neslab RTE-100 water bath. Cell path length of 1 mm was used. The spectra were recorded at a scan speed of 100 nm/min with an automatic slit width and 0.25-s response time. Ten spectra were collected for each sample, averaged, and corrected by subtraction of buffer spectrum.

Western Blotting

Proteins separated by SDS-PAGE gels (15%) were electroblotted onto nitrocellulose Hybond C-extra membrane (Amersham Corp.) and reacted with a rabbit anti-protease polyclonal serum, and antigen-antibody complexes were detected with protein A labeled with I and autoradiography.

Proteinase Assay

Protease activity was measured by the cleavage of core protein pre-VII to VII as detected by SDS-PAGE followed by autoradiography. The substrate consisted of [S]methionine-labeled purified disrupted ts1-39 °C virions(8) . The reaction mixture (20 µl) contained 10 µl of substrate, 5 µl of enzyme, and buffer (20 mM phosphate, pH 7.5). Activity was also measured by means of a fluorescent substrate assay (Molecular Probes, Eugene, OR) as described before(9) . (^2)

Urea Gradient Gel Electrophoresis

Gels were made by photopolymerization of a linear 0-8 M urea (ultrapure from Schwarz/Mann) and a compensating 15-11% (w/v) gradient of acrylamide as described previously(11, 12) . The gels were pre-electrophoresed for 1 h at 20 mA, and then 60 µg of pure protease sample (pRSET-A vector) was loaded and run at 4 °C in 0.05 M Tris acetate buffer, pH 4 (0.05 M acetic acid titrated to pH 4 with Tris) by applying 10 mA/gel toward the cathode. When the marker dye had entered the gel several millimeters, the current was increased and kept constant at 20 mA/gel for 7 h. The gel was stained with silver nitrate.

Secondary Structure Prediction

Eleven adenovirus protease sequences based on the EMBL/Swiss-prot release 30.0 were submitted to the PHD automatic mail server (predictprotein@EMBL-Heidelberg.DE) for protein secondary structure prediction based on a homology and neural network-derived protocol(13, 14, 15, 16) .


RESULTS AND DISCUSSION

Protease Purification, Identification, and Activity

The wild type and mutant recombinant Ad2 proteases were purified to homogeneity (Fig. 1, panelA). The apparent differences in molecular weight are due to the 40 and 15 amino acids fused to the N terminus of the proteases by the pRSET and pLPV vectors, respectively. We used two vector systems and two purification protocols to minimize the effect of artifacts on the properties of the protease. The identity of the proteins was confirmed by staining with anti-protease serum (Fig. 1, panelB). Both enzymes cleaved the viral substrate protein preVII specifically to VII (Fig. 1, panelC). The ts1 enzyme was not temperature-sensitive in its enzyme activity in vitro, albeit its activity was somewhat less than that of the wt enzyme, regardless of the temperature. Furthermore, the additional amino acids fused to the N termini did not significantly change enzyme activity, as measured by the fluorescent peptide assay (Table 1). We report elsewhere that the temperature-sensitive defect is confined to virus-infected cells and is likely a protein trafficking phenotype(4) .


Figure 1: Purification, identification, and activity of wild type and ts1 protease. The proteases were expressed in two vector systems, purified, and subjected to SDS-PAGE. PanelA, purified proteases stained with silver nitrate; lanes 1 and 3 show wt expressed by pRSET-A and pLPV, and lanes2 and 4 show ts1 proteases expressed by pRSET-A and pLPV. PanelB, a Western blot of the above stained with anti-protease serum. Panel C, cleavage of viral core protein pre-VII to VII by wild type and ts1 proteases.





Protein Instability

In the course of these and other studies involving a third expression system, we have frequently observed a lower protein yield for ts1 than for wt protease. Western blotting of expression lysates showed comparable yields of wt and ts1 enzyme in the insoluble pellet but an approximate 5-fold reduction of ts1 in the supernatant. Losses at certain steps in purification were also disproportionately larger for ts1 than wt. In addition, after purification, ts1 tended to precipitate more rapidly than wt. These observations suggest that the ts1 mutation destabilizes the protein, rendering it less soluble.

Urea Gradient Gel

The structure of the adenovirus protease was investigated using urea gradient gels. The electrophoretic mobility of a protein in urea gradient gels depends on its conformation and is useful in studying transitions between different conformational states (11, 12) . Both the wild type and mutant proteins show a rapid equilibrium between the native and unfolded states (Fig. 2). This is indicated by a continuous band of protein through the unfolding transition and by obtaining the same pattern irrespective of whether the folded or unfolded form of the protein is applied to the gel (results not shown). At low urea concentration the native form of protease predominates while at the higher urea concentration the unfolded form predominates. At the midpoint of the transition the concentration of the native and unfolded form is equal, and the free energy change for unfolding is zero.


Figure 2: Urea gradient gel electrophoresis of wt and ts1 protease. 60 µg of each protease was loaded along the top of the gel, electrophoresed, and stained with silver nitrate.



It is interesting to note that our results indicate that the Ad2 protease consists of a single conformational domain. The reported x-ray model structures of other thiol proteases, such as papain, actinidin, and picornaviral 3C enzymes, indicate that they are composed of two domains(17, 18) . Multidomain proteins usually exhibit complex equilibrium folding transitions if the individual domains unfold independently(19) . The results of our urea-gradient gel experiments suggest that the three-dimensional structure of the Ad2 protease may be different from that of the aforementioned cysteine proteases.

The calculated free energies of folding were very similar, -2.3 kcal/mol for the wild type enzyme and -2.6 kcal/mol for the ts1 enzyme, respectively. These values are consistent with other reports indicating that the replacement of surface proline residues in T4 lysozyme and the repressor lead to a free energy change of 0.4-0.8 kcal/mol(20) . A proline residue restricts the conformation of the polypeptide backbone to a much greater extent than leucine and other nonbranched amino acid residues. A proline to leucine replacement would be expected to increase the backbone configuration entropy of unfolding of a protein and potentially decrease protein stability(21, 22) . Our investigation indicates that the free energy of folding is smaller for the wild type than the mutant protein. This is surprising since the proline to leucine replacement in ts1 would normally be expected to create a destabilizing effect due to increased backbone entropy and therefore result in a reduction in the free energy of folding for the less stable protein. As shown earlier, the mutant protein is more prone to precipitation. It is possible that the 0.3 kcal/mol is within experimental error and does not give a clear picture of the effect of the modification. Further investigations will have to be performed to clarify this observation.

Circular Dichroism

The limited solubility of the protease in aqueous buffer led to the use of low concentrations of protein in the CD spectroscopy investigation. The low protein concentrations truncated the range of the CD spectrum that could be covered in our investigation. The CD bands in the amide region of the spectra were very similar between the wild type and the ts1 proteins (Fig. 3). Both proteins show very little alpha-helical content, and their spectra resemble that of beta-sheets and random coils. This indicates that the secondary structure of the protease is mainly beta-sheet since it has been reported that the CD spectra of some completely beta-sheet proteins resemble the spectra of models for random coils(23) . Our results show that the adenovirus protease has less alpha-helical content than other thiol proteases such as papain and actinidin, which contain 27% helical content(17) . This is consistent with our urea gradient gel investigation showing that unlike papain, which is composed of two domains, the adenovirus protease appears to contain a single domain.


Figure 3: Far UV CD spectra of mutant and wild type adenovirus protease. Wild type enzyme at 33 °C (--) and 39 °C(- - -) and ts1 enzyme at 33 °C(--) and 39 °C (bullet-bullet) in 20 mM MES buffer, pH 7. Protein concentration for both enzymes was 1.35 mM.



We also tried to investigate the possibility that in contrast to the wild type protein, the ts1 mutant protein may experience a change in secondary structure at 39 °C as compared with when it is at 33 °C. The CD spectra of both the wild type and ts1 proteins show little difference between data collected at the permissive (33 °C) and non-permissive (39 °C) temperatures. These results indicate that the changes in the properties of the protein resulting from the P137L mutation in ts1 are very small and may be too subtle to be monitored in vitro when using reconstituted recombinant proteins.

Secondary Structure

Currently there is no structural information on the adenovirus protease other than that reported here. Protein secondary structure prediction by combining evolutionary information and profile-fed neural networks have achieved greater than 70% accuracy(13, 14) . We decided to test this technique by using 11 adenovirus protease sequences of human, bovine, murine, and canine adenoviruses to derive a secondary structure prediction by means of the PHD program(15) . This prediction gave overall values of 42% alpha-helix, 11% beta-sheet, and 47% loop (Fig. 4). We are unable at present to explain the reason for the discrepancy between the CD-predicted and the PHD-predicted alpha-helical content. The active site cysteine 104 and histidine 54 residues appear embedded in regions without a clearly ordered structure. In a comparison of five classical cysteine proteases (papain, cathepsins), it was noted that the active site cysteine residue of each protein is in an alpha-helix while the active site histidine is in a beta-sheet segment(24) . By contrast, in the chymotrypsin-like cysteine proteases these residues are outside clearly ordered secondary structures(18) . Normally, serine-like cysteine proteases contain a catalytic triad consisting of a cysteine, a histidine, and an aspartic acid (or glutamic acid); other cysteine proteases like papain achieve catalysis with a cysteine-histidine couple(25) . Based on the order in which the cysteine nucleophile and the general base are disposed from the N terminus, the adenovirus protease follows the pattern of the picornaviral 3C enzymes(26) . We have attempted to determine the third residue that may possibly form an active site triad in Ad2. Ongoing investigations in our laboratory have yet to lead us to the identity of the third component of the triad, which would most likely be a conserved glutamic acid residue. (^3)As noted above, unlike other cysteine proteases that contain two domains, our urea gradient gel investigation shows that the Ad2 protease consists of a single conformational domain. These results suggest that the Ad2 proteases may have a structure resembling 3C proteases but with the unique feature of a single domain.


Figure 4: Predicted secondary structure of the adenovirus protease. Homology-derived secondary structure prediction based on 11 adenovirus protease sequences. The multiple sequence alignment was generated by the GCG PILEUP program. Numbering is according to the adenovirus type 2 (H-2) sequence. SUB SEC, the probability of alpha-helix (H), beta-sheet (E), and loop (L) structures. SUB ACC, accessibility to solvent: e, exposed; b, buried. The conserved His-54, Cys-104, Cys-122, and Cys-126 residues are in boldfaceitalic. The ts1 mutation is at residue Pro-137.



What Is the Effect of the Proline to Leucine Mutation?

These investigations indicate that the proline to leucine mutation does not appear to cause a major structural change to the recombinant enzyme. We were unable to monitor the subtle differences in the structure or function of the protein that lead to the observed changes in encapsidation when the ts1 mutant protein is expressed in its normal host. This is consistent with the general prediction that substitutions of solvent-exposed amino acids on the surfaces of proteins have little effect on protein structure(22) . It is known, however, that the cis-trans isomerization of proline residues may have a profound effect on protein structure, rate of folding, and protease susceptibility (27) . Our observation that the ts1 recombinant protein is less soluble than the wild type protein indicates that the cis-trans isomerization of this residue may have an important effect on the structure of the protein. In the predicted model of the protein, the Pro-137 residue is shown to be at the beginning of a long exposed loop stretching to residue Pro-165. This 28-amino acid loop contains 6 prolines while the entire 204-amino acid protein contains only 13 proline residues. The replacement of proline 137 by leucine in ts1 could conceivably result in an extension of the predicted alpha-helix that is shown to stretch from residues 120 to 134 (Fig. 4). This is possible since a study of the normalized frequency of occurrence in an alpha-helix for each of the possible residues has shown that leucine is the third most likely residue to occur within an alpha-helix(28) . If this is true for the Ad2 protease, the proline 137 to leucine replacement in ts1 would alter the size, shape, and stability of the proline-rich region that stretches to residue Pro-165. In addition, the mutation from a hydrophilic to a hydrophobic residue may have a tendency to extend the buried alpha-helix (Fig. 4) as predicted from studies of globular proteins(29) .

Since it has been proposed that proline may serve as an important regulatory signal in determining the lifetime and degradation rates of an enzyme(27) , another possible function for the proline residue is as a pacemaker in the proper folding or trafficking of the protein during capsid formation. The location of Pro-137 in a predicted surface loop is consistent with such a function and could account for the large 4-log difference in the formation of infectious virions observed in vivo(10) .


FOOTNOTES

*
This research was supported by Grant MT4164 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a studentship from the government of Iran.

Chercheur-Boursier of the FRSQ. Present address: Dept. of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.

**
To whom correspondence should be addressed: Dept. of Microbiologie, Faculte de Medecine, Universite de Sherbrooke, 3001, 12 Ave. N., Sherbrooke, Quebec J1H 5N4, Canada. Fax: 819-564-5392; j.weber{at}courrier.usherb.ca.

(^1)
The abbreviations used are: Ad2, adenovirus type 2; wt, wild type; MCAC, metal chelating affinity chromatography; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.

(^2)
Diouri, M., Geoghegan, K. F., and Weber, J. M. Protein and Peptide Lett., in press.

(^3)
C. Rancourt, H. Keyvani-Amineh, M. Diouri, and J. M. Weber, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Professor N. Voyer of the chemistry department for the use of the spectropolarimeter.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.