(Received for publication, September 7, 1995; and in revised form, December 8, 1995)
From the
Recently, the mechanism of autoprocessing of the protease (PR)
of the human immunodeficiency virus type 1 from the model polyprotein,
MBP-TF-PR-
Pol, which contains the protease linked to short
native flanking sequences (
TF and
Pol) fused to the maltose
binding protein (MBP) of Escherichia coli, was reported
(Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R., and Jerina,
D. M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91,
7970-7974). According to this mechanism, intramolecular cleavage
of the N-terminal strands of the dimeric MBP-
TF-PR-
Pol
protein leads to the formation of the PR-
Pol intermediate, which
is subsequently converted to the mature protease by cleavage of the
C-terminal strands. We now report the purification and characterization
of the PR-
Pol intermediate and the kinetics of its processing to
the mature protease. Unlike the MBP-
TF-PR-
Pol precursor,
PR-
Pol has proteolytic activity similar to that of the mature
enzyme at pH 5.0. The pH rate profile for k
/K
is similar to
that of the mature protease above pH 4.0. Although the PR-
Pol is
more sensitive than the mature protease toward denaturing reagents,
both the enzymatic activity and the intrinsic fluorescence of
PR-
Pol are linearly dependent on the protein concentration,
indicating that the protein is largely in its dimeric form above 10
nM. In contrast to the first-order kinetics observed for the
proteolytic reaction at the N terminus of the protease, the proteolytic
reaction at the C terminus of the protease is second order in protein
concentration. These results are discussed in terms of a mechanism in
which the C-terminally located
Pol peptide chains are cleaved
intermolecularly to release the mature protease.
Limited proteolysis by specific proteases is an established
post-translational mechanism by which cellular and viral precursor
proteins are converted to functional
species(1, 2, 3) . Retroviruses, including
the human immunodeficiency virus type 1 (HIV-1), ()encode a
protease that is a member of the family of aspartic acid
proteases(1, 2, 3) . The HIV-1 protease is
translated as a part of the Gag-Pol polyprotein flanked by the
transframe (TF, also termed p6*) and reverse transcriptase proteins at
its N and C termini,
respectively(1, 2, 3, 4) . The
protease is responsible for its own maturation and for the release of
structural and active functional proteins required for viral
replication(1, 2, 3) . The mature protease is
a symmetrical homodimer whose active site is formed along the interface
between the two subunits with each subunit contributing one of the two
catalytically important aspartic acid residues(5) . Partial
inhibition of the protease in infected cells(6) , premature
processing of viral polyproteins(7) , or overexpression of the
Gag-Pol polyprotein relative to the Gag precursor (8) results
in aberrant virus assembly and formation of non-infectious particles.
In addition, activation of the protease is initiated on the membrane of
infected cells(9) . These results suggest that temporal and
spatial regulation of protease activity during the viral life cycle is
crucial for proper assembly and maturation of the viral polyproteins to
produce infectious particles.
Since studies of the autoprocessing
reaction of the Gag-Pol polyprotein are complicated by the presence of
multiple cleavage sites(1) , we developed a model protease
precursor that contains only two cleavage sites. This model polyprotein
(MBP-TF-PR-
Pol) was constructed by the addition of 19
residues of the native reverse transcriptase sequence (
Pol) to the
C terminus of the protease domain and 12 residues of the native
transframe region (
TF) to its N terminus, which was additionally
fused to the maltose binding protein (MBP) of Escherichia
coli(10) . Specific mutational analyses of the two
cleavage sites in the model polyprotein showed that the N-terminal
cleavage precedes the C-terminal cleavage and that the N-terminal
cleavage site is more sensitive to mutations(11) . These
studies also showed that blocking the N-terminal cleavage further
restricts cleavage at the C terminus of the protease(10) .
Investigation of the autoprocessing of the model polyprotein led to the
mechanism summarized in step 1 of Fig. 1(12) .
Initially, the protease domain of the model polyprotein forms a dimer
with an active site that is capable of binding known substrates and
inhibitors of the mature protease. The dimeric polyprotein, which has
low intrinsic catalytic activity, is converted to the mature protease
in two steps. The first step includes intramolecular cleavage at the N
terminus of the protease to produce the PR-
Pol intermediate, which
is subsequently converted to the mature protease by cleavage of the
C-terminal strands in the second step. In this study, we describe (i)
purification of the transient PR-
Pol, (ii) characterization of the
catalytic activity of PR-
Pol and its stability toward denaturing
reagents in comparison with the mature protease, and (iii) kinetics of
proteolytic processing of PR-
Pol to mature protease. Finally, the
results presented are discussed in terms of a mechanism in which the
hydrolysis of the
Pol sequence, step 2 (Fig. 1), occurs via
an intermolecular process.
Figure 1:
Proposed mechanism for the
autoprocessing of HIV-1 protease from the model polyprotein
MBP-TF-PR-
Pol (12) . The MBP and protease are denoted
as large hatched ovals and small closed ovals,
respectively. Lines represent
TF and
Pol sequences
that flank the protease domain. The protease catalyzes the hydrolysis
at its N terminus from a dimeric MBP-
TF-PR-
Pol via an
intramolecular mechanism (12) to release the intermediate
PR-
Pol (step 1). Results presented in this paper show
that the conversion of the PR-
Pol to the mature protease occurs
via an intermolecular process (step
2).
Buffers employed in this study were buffer A (50 mM Tris-HCl at pH 8.2, 25 mM NaCl, 1 mM EDTA, and
0.1 M DTT), buffer B (100 mM sodium acetate at pH
5.1, 1 mM DTT, 1 mM EDTA, 0.05% Triton X-100, 1.6%
MeSO, and 0.8 µM pepstatin A), buffer C (50
mM Tris-HCl at pH 8.2, 5 M urea, 5 mM DTT, 5
mM EDTA, and 0.5% Tween 20), and buffer D (100 mM sodium acetate at pH 5.0, 1 mM DTT, 1 mM EDTA,
and 0.05% reduced Triton X-100). Unless otherwise indicated, all
incubations, assays, and measurements were carried out at 25 °C.
At low
enzyme concentrations, a fluorometric assay was used(14) . In a
typical measurement, 135 µl of buffer D, which was 9 µM in peptide II,
4-(4-dimethylaminophenylazo)benzoyl--aminobutyryl-Ser-Gln-AsnTyr-Pro-Ile-Val-Gln-[(2-aminoethyl)amino]-naphthalene-1-sulfonic
acid (Bachem Bioscience Inc., PA), was placed in a microfluorescence
cell (Hellma Cells Inc., NY). Reaction was initiated by adding 15
µl of the protein solution in buffer C and monitored by following
the increase in fluorescence at 490 nm using an excitation wavelength
of 340 nm.
Figure 2:
Purification of PR-Pol by gel
filtration chromatography. Processing of the protease from the purified
MBP-
TF-PR-
Pol was initiated by renaturation of the protein in
the presence of pepstatin A. After 2.5 h, the reaction was terminated
by precipitation, and proteins were collected by centrifugation and
fractionated under denaturing conditions. A, elution monitored
for changes in absorbance at 280 nm (closed circles) and
protease activity (open circles). B, fractions
3-8 were subjected to SDS-PAGE on Tris-Tricine gels, and proteins
were visualized by Coomassie staining. The MBP-
TF-PR-
Pol,
MBP-
TF, and PR-
Pol are indicated as 52-, 38-, and 13.2-kDa
proteins, respectively. The mature protease elutes in fractions 9 and
10 under the same conditions.
Figure 3:
pH dependence of k/K
for
PR-
Pol-catalyzed hydrolysis of peptide I in Mes, acetate, and
formate buffers at 25 °C.
Figure 4:
Urea denaturation curves for PR-Pol (open circles) and protease (filled circles) as
determined by measuring changes in enzymatic activity in buffer D
without Triton X-100 at 25 °C.
The
intrinsic fluorescence of the PR-Pol and the mature protease
displays a sigmoidal decrease in intensity with the lowering of pH. The
fluorescence changes occur over a narrow pH range. For PR-
Pol, the
range is
0.4 pH unit with an inflection at pH 3.7, whereas for the
mature protease the changes occur over a range of 0.9 pH unit with an
inflection point at pH 2.9. Fluorescence spectra obtained at pH 2.5 and
>4 for both proteins are identical to those of the urea-denatured
and enzymatically active proteins, respectively. Similar fluorescence
changes are observed on acid denaturation of staphylococcal nuclease (15) . Thus, the pH-dependent fluorescence change of
PR-
Pol and mature protease is due to protein denaturation.
Figure 5:
Time course of the conversion of
PR-Pol to protease monitored by immunoblotting analysis. Aliquots
were drawn at the indicated times, and proteins were separated by
SDS-PAGE, transferred to nitrocellulose membrane, and probed with a
protease specific antibody.
Figure 6:
Kinetics of the conversion of PR-Pol
to protease at pH 5.0 in buffer C at 25 °C. The reactions were
monitored by immunoblotting, and band intensities corresponding to
PR-
Pol and protease were quantified by densitometry. Reactions
were monitored to
25% conversion, and initial rates were estimated
from plots of conversion versus time. Band intensities are
linearly dependent on the amount of protein blotted as shown in inset.
We have developed a procedure to purify the protein
intermediate PR-Pol (see Fig. 1) to near homogeneity and
examined the mechanism of the C-terminal proteolytic cleavage of HIV-1
protease from MBP-
TF-PR-
Pol. The method described, which
requires addition of pepstatin A in the autoprocessing reaction, allows
the isolation in 18% yield of the PR-
Pol formed during the
autoprocessing of MBP-
TF-PR-
Pol and substantially increases
the accumulation of PR-
Pol relative to mature protease. In
addition, since PR-
Pol and the mature protease were separable by
gel filtration chromatography under denaturing conditions, the purified
PR-
Pol was shown to contain very little mature protease.
Both
protease and PR-Pol fold and dimerize instantaneously with
concomitant appearance of enzymatic activity after the denatured
proteins are diluted from 5 M urea with 10 volumes of acetate
buffer at pH 5.0. The proteolytic activity of the PR-
Pol is nearly
indistinguishable from that of the mature protease at pH 5.0 (Table 1) as is the pH rate profile for k
/K
for the
PR-
Pol-catalyzed hydrolysis of peptide I above pH 4.0. The
pK
of about 5.2 determined from the pH rate
profile of PR-
Pol is in agreement with the pK
of 4.8 obtained for the mature protease(16) . These
results clearly demonstrate that the addition of C-terminal flanking
sequences to the protease does not distort the active site of the
enzyme nor alter the environment of the catalytic groups.
Unlike the
cleavage of the protease at its N terminus, which is an intramolecular
reaction of the dimeric fusion protein and follows first-order
kinetics(12) , the cleavage at its C terminus is second order
in protein concentration (Fig. 6). Since PR-Pol is
predominantly in the dimeric form at a concentration
10 nM (see below), the most reasonable explanation for the second-order
kinetics is that the cleavage of
Pol occurs via an intermolecular
mechanism (Fig. 1). In such a mechanism, a PR-
Pol dimer
catalyzes the hydrolysis of the C-terminal
Pol sequences of
another dimer with an observed second-order rate constant of 675
± 30 M
s
(100
mM acetate, pH 5.0, at 25 °C), which corresponds to k
/K
for this reaction.
The above mechanism is somewhat unexpected since the structure of
the dimeric mature protease shows that the C-terminal strand of one
subunit is involved in a hydrogen bond network with the N- and
C-terminal strands of the other subunit (see below). Therefore, the
C-terminal strands of a dimeric PR-Pol may not be easily
accessible to bind to another enzyme dimer and that should be reflected
in the observed second-order rate constant. The k
/K
for the HIV-1
protease-catalyzed hydrolysis of a peptide substrate spanning the
C-terminal cleavage site is 24,000 M
s
in 0.2 M phosphate buffer, pH 5.6,
and 2 M NaCl at 37 °C(17) . It is unlikely that
the large (
40-fold) difference in k
/K
for this substrate
relative to cleavage of PR-
Pol results only from differences in
the experimental conditions. The value of k
/K
increases with a higher
salt concentration (18, 19) and temperature and
decreases with a higher pH(17) . Thus, the large difference in k
/K
must be due to the
inaccessibility of the C-terminal sequence in the dimeric structure of
PR-
Pol.
Attachment of short native sequences to the N or the C
termini of the protease domain of HIV-1 has little effect, if any, on
the proteolytic activity, whereas non-native and/or long flanking
sequences decrease the enzymatic activity. Tang and co-workers (20) compared the proteolytic activity of a mutant protease
(A28S) with and without 25 amino acids of the native TF region linked
at its N terminus. These two proteins were shown to have similar
enzymatic activities. Sequence alignment of various retroviral
proteases show that several of these enzymes have short sequences that
extend beyond the HIV-1 protease (21) . These results are
consistent with the observed catalytic activity of PR-Pol. In
contrast, the attachment of the non-native 14-kDa protein A sequence at
the N terminus of HIV-1 protease causes a 10-fold decrease in catalytic
activity(22) . MBP-
TF-PR-
Pol, which contains the
38-kDa MBP domain, has at least 600-fold lower catalytic activity than
the mature protease(12) . Qualitatively, these results indicate
that the type of sequence and/or its length have an influence on the
proteolytic activity.
The major difference observed between
PR-Pol and the mature protease is in their conformational
stabilities. PR-
Pol is more sensitive toward urea and acid
denaturation than the mature protease. These results can be explained
based on the three-dimensional structure of the mature protease. The
major interactions between the two subunits of the mature protease
occur in the region of the N- and C-terminal strands. The two
C-terminal strands form an antiparallel
-sheet, and the N terminus
of each subunit interacts with the C terminus of the other subunit to
extend the antiparallel
-sheet(5) . Each positively
charged N terminus is anchored to the
-sheet by an electrostatic
interaction with the negatively charged carboxylate group of the C
terminus(23) . Since the dimeric form of PR-
Pol lacks this
electrostatic interaction, it is expected to be less stable than the
mature protease in urea. It should be pointed out that the
Pol
sequences may provide some additional interactions between the two
subunits to partially compensate for the absence of electrostatic
interactions.
Although the addition of the Pol sequence at the
C terminus of the protease destabilizes PR-
Pol, the results
presented in this study show no evidence that it alters the
monomer-dimer equilibrium. Above 10 nM, both the rate of
PR-
Pol-catalyzed hydrolysis of peptide II and the measured
intrinsic protein fluorescence are linearly dependent on the protein
concentration. Due to the sensitivity limits of the protease assay
using peptide II and the intrinsic fluorescence measurement, the
monomer-dimer equilibrium of PR-
Pol could not be assessed below 10
nM. Since enzymatic activity requires dimer formation, these
results indicate that PR-
Pol is predominantly dimeric under our
experimental conditions.
In vitro studies of the
autoprocessing of HIV-1 protease from the model polyprotein
MBP-TF-PR-
Pol provide a plausible mechanism for the
maturation of the viral Gag-Pol polyprotein similar to that in Fig. 1. Our previous results suggested that the dimeric Gag-Pol
polyprotein has low intrinsic proteolytic activity relative to that of
the mature enzyme and that the cleavage at the N terminus of the
protease domain occurs via an intramolecular mechanism(12) .
This mechanism for the separation of the structural and functional
proteins is consistent with proteolytic steps involved in the
processing of other viral polyproteins(2) . The low proteolytic
activity of the dimeric Gag-Pol may be necessary to prevent the
separation of the functional domain that contains the protease, reverse
transcriptase, and integrase until the assembly of the viral particle
is complete. On the other hand, the Pol product resulting from the
initial cleavage may possess enzymatic activity comparable to that of
the mature protease, which allows it to carry out the remainder of the
processing events leading to the release of the mature proteins via
intermolecular reactions.