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 (
)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
, 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
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
-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
-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
-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
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) . (
)
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
-helical content,
and their spectra resemble that of
-sheets and random coils. This
indicates that the secondary structure of the protease is mainly
-sheet since it has been reported that the CD spectra of some
completely
-sheet proteins resemble the spectra of models for
random coils(23) . Our results show that the adenovirus
protease has less
-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
(
-
) 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%
-helix, 11%
-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
-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
-helix while the active site
histidine is in a
-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. (
)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
-helix (H),
-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
-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
-helix for
each of the possible residues has shown that leucine is the third most
likely residue to occur within an
-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
-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) .