(Received for publication, July 27, 1995; and in revised form, September 20, 1995)
From the
The differences in substrate specificity between Moloney murine
leukemia virus protease (MuLV PR) and human immunodeficiency virus
(HIV) PR were investigated by site-directed mutagenesis. Various amino
acids, which are predicted to form the substrate binding site of MuLV
PR, were replaced by the equivalent ones in HIV-1 and HIV-2 PRs. The
expressed mutants were assayed with the substrate
Val-Ser-Gln-Asn-TyrPro-Ile-Val-Gln-NH
(
indicates the cleavage site) and a series of analogs containing single
amino acid substitutions in positions P
(Ser) to
P
`(Val). Mutations at the predicted
S
/S
` subsites of MuLV PR have a strong
influence on the substrate specificity of this enzyme, as observed with
mutants H37D, V39I, V54I, A57I, and L92I. On the other hand,
substitutions at the flap region of MuLV PR often rendered enzymes with
low activity (e.g. W53I/Q55G). Three amino acids (His-37,
Val-39, and Ala-57) were identified as the major determinants of the
differences in substrate specificity between MuLV and HIV PRs.
Retroviral maturation involves the proteolytic cleavage of viral
precursor polyproteins by an aspartyl protease (PR) ()encoded within the virus genome(1) . Since the
discovery of the human immunodeficiency virus (HIV) as an etiological
agent causing the acquired immunodeficiency syndrome (AIDS), these
proteases have been widely studied as potential targets of antiviral
drugs. As a result, many PR inhibitors have been synthesized and a
number of them are currently undergoing in vitro screening or
clinical evaluation(2, 3) .
Retroviral PRs are
homodimeric enzymes(4) . Each subunit contains the conserved
sequence Asp-Thr(Ser)-Gly, which provides the aspartyl group necessary
for catalysis. Crystal structures of retroviral PRs in their native
form and complexed with inhibitors have been
determined(2, 5, 6, 7, 8, 9, 10) .
These studies have shown that the substrate is bound to the PR in an
extended conformation, maintained by hydrogen bond interactions
between PR residues and the amide and carbonyl groups of the peptidic
substrate. The PR dimer forms a series of subsites (termed
S
, S
, S
, S
,
S
`, S
`, and S
`), which correspond
to the binding sites of the P
, P
,
P
, P
, P
`, P
`, and
P
` residues of the substrate, where the scissile bond is
located between the P
and P
` positions.
Oligopeptide substrates are useful tools to define the specific
requirements of the binding site and therefore to analyze the substrate
specificity of the retroviral PRs. The model peptide VSQNY
PIVQ
(
indicates the cleavage site), which derives from the MA/CA
processing site in HIV-1 Gag, and a series of analogs containing single
amino acid substitutions at P
to P
` positions
have been used previously to compare the substrate specificity of
various retroviral
PRs(11, 12, 13, 14) . The MuLV PR
has a strong preference for analogs with hydrophobic residues, such as
Val or Ile at P
, and Ile or Leu at P
, in
contrast to HIV-1 and HIV-2 PRs, which prefer smaller or more polar
residues at both positions(14) . The amino acid sequences of
MuLV and HIV-1 PR share 27% identical residues (Fig. 1). A
molecular model of MuLV PR was built on the basis of the crystal
structure of HIV-1 PR(14) . Although the general topology of
the MuLV and HIV PRs was predicted to be very similar, only 6 of the 22
residues found in subsites S
to S
` of MuLV PR
are conserved in both HIV PRs, based on our proposed model. The
differences in specificity between both enzymes were attributed to the
greater hydrophobicity of the S
subsite and the larger size
of the S
subsite in the MuLV PR.
Figure 1:
Sequence comparison of Moloney MuLV,
HIV-1, and HIV-2
PRs. Amino acids, which
are identical in MuLV PR and either HIV-1 or HIV-2 PRs, are boxed. The elements of secondary structure observed for the
crystal structure of HIV-1 PR are indicated by letters a through d, a` through d`, and q for
-strands, and h` for the only
-helical
segment of the PR monomer(5) . Residues 43-58 in HIV-1 PR
form the flap, which includes
-strand a`, part of
-strand b`,
and the residues between both
-structures. The MuLV PR amino acid
sequence was taken from Yoshinaka et al.(15) , and the
HIV PR sequences were from Copeland and
Oroszlan(16) .
In this paper,
site-directed mutagenesis has been used to identify critical residues
which determine the substrate specificity of MuLV PR. Amino acids
forming the MuLV PR subsites were replaced by the equivalent residues
found in HIV-1 and HIV-2 PRs. The mutant PRs were assayed for
proteolytic activity using VSQNYPIVQ analogs. The results of this
analysis indicate that residues in subsites
S/S
` are important for substrate specificity,
while the flap region is very sensitive to mutations. We identified
His-37, Val-39, and Ala-57 as the major determinants of the differences
in substrate specificity between MuLV and HIV PRs.
Recombinant HIV-1 PR was prepared from E. coli inclusion bodies according to previously published procedures(20) . E. coli strain BL12(DE3)pLysS harboring the plasmid pET-HIVPR (21) was kindly provided by Dr. Jordan Tang, Oklahoma Medical Research Foundation.
The comparison of the crystal structures of HIV PR-inhibitor
complexes with the molecular model of Moloney MuLV PR revealed many
amino acid differences at the substrate binding sites of both enzymes (Fig. 2). Only 6 of the 22 residues found in subsites S to S
` of MuLV PR (Leu-30, Asp-32, Gly-34, Ala-35,
Gly-56, and Pro-89) are conserved in both HIV-1 and HIV-2 PRs. The
remaining nonconserved 16 amino acids, which are predicted to be part
of MuLV PR subsites, are scattered throughout the substrate binding
pocket. None of the PR subsites is conserved in the three enzymes.
Site-directed mutagenesis was used to generate a large number of
mutants having single amino acid substitutions at the substrate binding
pockets of MuLV PR. All the amino acids which were predicted to be part
of MuLV PR subsites were systematically replaced by those found in the
equivalent positions of HIV-1 or HIV-2 PRs. Enzymatic characterization
of these mutants was limited to those that could be expressed and
purified in significant amounts. Approximately half of the constructs
were excluded from the analysis, since they rendered proteins which
were poorly expressed, insoluble, or not stable (data not shown).
Mutated MuLV PRs with single or double amino acid replacements at each
of the PR subsites (S
to S
`) were then used to
investigate the molecular basis of the substrate specificity
differences between MuLV and HIV PRs. The introduced mutations were
located in both subunits due to the homodimeric nature of MuLV PR. The
activity of the PR mutants was tested by using the peptide VSQNYPIVQ
and a series of substrate analogs having substitutions at the P
to P
` positions.
Figure 2:
Residues forming the substrate binding
pocket of MuLV and HIV PRs. Left, schematic representation of
the HIV-1 MA/CA substrate, VSQNYPIVQ (one-letter amino acid code), from
P to P
` in the S
to S
`
subsites of MuLV PR. Primes indicate residues in the second
subunit of the dimer. The relative size of each subsite is illustrated
by the area enclosed by the curved line around each substrate
side chain. Only protease residues that differ between MuLV and HIV PRs
are shown. At each subsite the MuLV PR residue and HIV-1 PR residue are
shown outside and inside the parentheses, respectively. HIV-2
PR residues are underlined and also inside the parentheses. Right, diagram showing the contribution
of residues of the substrate binding pocket to subsites S
to S
`. Equivalent residues in MuLV and HIV PRs that
contribute to the same subsite are shown in black. Residues
contributing only to MuLV PR subsites are horizontally dashed,
while those contributing only to HIV PR subsites are vertically
dashed.
The catalytic parameters (k and K
) for the cleavage
of VSQNYPIVQ by the wild type MuLV PR and 14 mutant enzymes are given
in Table 2. It should be noted that the wild type PR, as well as
the mutant enzymes were expressed in fusion with the S. japonicum glutathione S-transferase and then excised with thrombin
to release the PR, which contained two extra amino acids (Gly-Ser-) at
the amino-terminal sequence, not found in the viral protease isolated
from purified virus(15) . These residues are required to
maintain the integrity of the thrombin cleavage site. The influence of
the amino-terminal extension on the proteolytic activity is apparently
negligible. Thus, a mutated MuLV PR (NT), that lacks its natural
amino-terminal extension TLDD (see Fig. 1), was found to cleave
the reference oligopeptide substrate with similar k
and K
values to those of the wild type
enzyme (Table 2). Similar kinetic parameters were also observed
for mutants E15R, V39I, V54I, and for the double mutant V39I/V54I. The
mutant H37D shows a significant decrease of the K
value. The double mutant W53I/Q55G, which involves amino acids
located at the flap region of the PR, did not cleave the oligopeptide
VSQNYPIVQ and showed negligible activity when the analogs
P
(Leu) or P
(Ile) were used as substrates (k
< 0.001 s
). Other
replacements involving residues at the flap (e.g. W53I or
G60F) rendered PRs showing a decreased affinity for the substrate. The
flap region is very sensitive to changes which can alter the catalytic
rate, as well as the specificity, as shown for other retroviral PRs,
such as HIV-1 or Rous sarcoma virus
PRs(20, 24, 25, 26) . Mutations C88T
and L92I, which affect subsites S
-S
`, also
rendered PRs with a low catalytic efficiency for this substrate.
All
MuLV PR mutants were examined for changes in substrate preference using
a series of VSQNYPIVQ analogs with substitutions in the P to P
` positions. The proteolytic activity obtained
using the substituted analogs relative to that observed with VSQNYPIVQ
was determined for all mutant PRs and compared with the wild type MuLV
and HIV-1 PRs (Fig. 3). Large differences in substrate
specificity were detected with analogs having large hydrophobic amino
acids at P
and P
positions. Less variation was
produced by the substitutions tested at P
, P
,
and P
`. The PR mutants can be classified into three
categories, based on the results shown in Fig. 3. First, NT,
E15R, and Y63V appear to be similar to the wild type MuLV PR. According
to the MuLV PR model, the amino-terminal residues are predicted to be
distant from the substrate binding site and are not part of the
structure that is conserved in HIV-1 PR. The side chain of Glu-15 forms
ionic interactions with either Arg-17 or Arg-95 which would be
eliminated in the E15R mutant. Since Glu-15 is located near P
and P
`, we would predict that substitution E15R would
have an effect on P
- and P
`-substituted
substrates. However, no effect was observed, probably because only
conservative substitutions of P
and P
` were
tested. Tyr-63 is predicted to lie on the flap near P
and
P
. Tyr-63 is an aromatic residue with a polar hydroxyl
group and lies near His-84, suggesting that the Y63V substitution may
have indirect effects through His-84, which is closer to the substrate.
The mutant Y63V showed a slight preference for hydrophobic residues at
P
, perhaps due to the absence of the polar hydroxyl group
of Tyr and fewer interactions with His-84. A second group of mutants
includes W53I, V54I, G60F, C88T, and L92I. The introduction of these
mutations in the MuLV PR resulted in an increased preference for
hydrophobic amino acids at P
and P
positions in
VSQNYPIVQ. In the wild type MuLV PR, the catalytic efficiency was
6-fold higher with P
(Leu) and P
(Ile) than with
VSQNYPIVQ (Table 3). This increase was even more pronounced for
the mutant enzymes of this group. For example, the k
/K
value obtained for L92I
and VSQNYPIVQ was 0.03 mM
s
versus 4.59 mM
s
, obtained with the P
(Leu) analog.
Trp-53, Val-54, and Gly-60 are located in the flap region of the PR. In
the model of MuLV PR, Trp-53 is predicted to lie close to P
and would be expected to make the S
subsite smaller
than in HIV PRs. Accordingly, the substitution of Trp-53 by Ile favors
the cleavage of analogs having larger residues at P
(Fig. 3). Trp-53 is not predicted to be close to P
,
unless it can insert into the PR structure in a very different
conformation than the equivalent Met-46 of HIV-1 PR. The mutant V54I is
predicted to reduce the size and increase the hydrophobicity of
subsites S
, S
, and S
` so that
smaller, more hydrophobic residues at P
, P
, and
P
` will form better substrates, as observed in our assays.
Gly-60 in MuLV PR is equivalent to Phe-53 in HIV PR which lies across
the two antiparallel flap strands. The substitution of Gly by the
hydrophobic Phe, as in G60F, would increase the hydrophobicity of the
S
/S
` and S
/S
` subsites,
consistent with the observed effects with P
- and
P
-substituted analogs. In addition, MuLV PR Gly-60 is close
to P
, but would be predicted to have an indirect effect
since Gln-55 lies between Gly-60 and P
. Mutant G60F showed
reduced cleavage of P
(Asn). C88T and L92I are predicted to
influence binding at S
, S
`, S
, and
S
` subsites ( Fig. 2and Fig. 3). The mutation
C88T introduces a more bulky side chain that may increase the
preference for hydrophobic residues at P
, as observed. In a
similar way, mutant L92I involves the introduction of a more bulky
residue which probably explains the preference for the smaller
P
(Met) over the larger Tyr and the increased selection of
more hydrophobic residues at P
. Finally, a third group of
mutants appears to be responsible for the major differences in
substrate specificity between MuLV and HIV PRs. These mutations include
H37D, V39I, A57I, and the double mutant V39I/A57I. In the model
structure of MuLV PR, His-37 is close to P
and P
and is predicted to form a hydrogen bond interaction with Gln-36.
In the crystal structures of HIV-1 PR, a hydrogen bond is formed
between Asp-30 (His-37 equivalent) and Asn-88 (Asp-96 equivalent). The
interaction seen in HIV PR and modeled for MuLV PR cannot occur in the
H37D mutant, because the acidic Asp-37 would repel residue Asp-96 and
instead may form an ion pair with His-84. H37D can still form a
hydrogen bond interaction with Ser at P
of the substrate
and will probably decrease the hydrophobicity of S
,
S
, and S
` subsites, giving rise to the observed
preference for less hydrophobic residues at P
,
P
, and P
`. Despite the observed changes in
specificity in the S
subsite using the H37D mutant, the
substitution of His-37 by Asp was not sufficient to obtain cleavage of
the P
(Asp) analog. This oligopeptide was cleaved by the
HIV-1 PR (relative activity: 0.23), but it was a poor substrate for
MuLV PR, as well as for all the mutants tested (relative activity <
0.01). The mutant V39I is predicted to reduce the space available in
S
and S
` due to the presence of the larger Ile
rather than Val. The presence of the larger Ile-39 is consistent with
the decreased activity for substrates with the longer Leu at P
and P
`. The shorter but more bulky Ile at P
can still fit in the subsite because of the presence of Leu-92 at
the side of the subsite, rather than the more bulky Ile-84 as in HIV-1
PR. The mutation A57I lies at the tip of the flaps near substrate
positions P
, P
, P
`,and
P
`. The presence of the larger Ile is expected to reduce
the size of the affected subsites, in agreement with the observed poor
cleavage of the larger hydrophobic residues at P
. The
double mutant V39I/A57I shows the simultaneous effect of both mutations
in reducing the size of subsites S
and S
`.
Substrate analogs having Leu, Ile,or Val at P
are cleaved
much less efficiently by V39I/A57I than by the single-amino acid
mutants V39I or A57I. In addition, the oligopeptide having Ala at
P
` position was not cleaved by the wild-type MuLV PR and by
most of the mutants tested. In contrast, we observed cleavage of this
peptide when mutants A57I and V39I/A57I, or the HIV-1 PR,were used in
the assays. In combination with the V54I mutation, V39I can also
attenuate the preference for Leu over Asn at P
position
shown by the V54I mutant (Fig. 3). This effect can be related to
changes in the catalytic parameters. For example, the K
value for the oligopeptide analog P
(Leu) is
significantly higher for mutants V39I and V39I/V54I than for V54I (Table 3).
Figure 3:
Protease activities with VSQNYPIVQ analogs
with substitutions at P, P
, P
,
P
, P
`, and P
` positions. For each
enzyme, the data are expressed as activity with the modified substrates
relative to that observed with the reference peptide (VSQNY
PIVQ).
The following peptides were used: P
(Ile), VIQNY
PIVQ;
P
(Thr), VTQNY
PIVQ; P
(Ala),
VAQNY
PIVQ; P
(Asn), VSNNY
PIVQ;
P
(Leu), VSQLY
PIVQ; P
(Ile),
VSQIY
PIVQ; P
(Val), VSQVY
PIVQ;
P
(Ala), VSQAY
PIVQ; P
(Met),
VSQNM
PIVQ; P
`(Leu), VSQNY
PLVQ; and
P
`(Ile), VSQNY
PIIQ. The
indicates the protease
cleavage site. The substituted amino acids are shown in bold.
Deviations between duplicates were always lower than 20%. Note that the
scales for relative activity are different depending on the
substrate.
Previously, the comparison of the proteolytic activities of
wild type MuLV and HIV PRs using a series of VSQNYPIVQ analogs led us
to conclude that the MuLV PR showed a stronger preference for
oligopeptides having larger hydrophobic residues at P and
P
positions(14) . The results of our mutational
studies have now revealed that these differences in substrate
specificity were mainly caused by amino acid changes at the S
subsite of the PR. The substitution of His-37, Val-39,or Ala-57
in MuLV PR by the equivalent residues found in HIV PRs (Asp-30,
Ile-32,or Ile-50) rendered proteolytic enzymes with a diminished
preference for hydrophobic residues at P
. Similar effects
of mutating His-37 were also observed with P
-substituted
analogs. Interestingly, the mutant H37D showed a higher affinity for
the substrate than the wild-type PR. This is probably due to the
formation of a hydrogen bond interaction with the hydroxyl group of
serine at the P
position of the substrate, as previously
predicted for HIV PRs(11) . In Rous sarcoma virus PR, the
equivalent residues of MuLV PR His-37 and Val-39 were shown to be
critical for selection of the P
/P
and
P
/P
` substrate positions (26) . Our
results are also consistent with the notion that the S
subsite exerts the greatest influence in substrate selection in
HIV-1 PR, due to its relatively small size and its major contribution
to the hydrogen bond network maintaining PR-substrate
interactions(27) . Development of viral resistance to various
PR inhibitors is often associated with mutations involving amino acids
forming the S
/S
` subsites of the HIV-1 PR (e.g. V32I(28, 29) , I47V(30) ,
G48V(29, 31) , I50V (29, 30) , and
I84V(29, 30, 32, 33, 34, 35) ).
Furthermore, differences in specificity between HIV-1 and HIV-2 PRs
have been ascribed to the presence of Val-32 and Ile-47 in the S
subsite of HIV-1 PR, compared with Ile-32 and Val-47 in HIV-2
PR(12, 36) .
Analysis of the molecular model of
MuLV PR suggests that the preference for larger hydrophobic residues at
P can be correlated with the larger size of the S
subsite of MuLV PR, compared with HIV PRs. The smaller Val-39
rather than Ile at the top of the subsite allows longer hydrophobic
residues like Leu to fit at P
and P
`. In a
similar way, the presence of the larger Ile in the mutant A57I is
expected to reduce the size of S
and S
`
subsites. Accordingly, VSQNYPIVQ analogs with larger hydrophobic
residues at P
are less efficiently cleaved by mutant A57I
than by the wild type MuLV PR, and analogs having Ala at P
`
become better substrates of the mutant PR. These effects are even more
pronounced in the double mutant V39I/A57I, whose catalytic efficiency
with the P
(Leu) analog is about 10-20 times lower
than with P
(Ile) or VSQNYPIVQ. Interestingly, differences
in specificity between the equine infectious anemia virus PR and the
HIV-1 PR have been attributed to Thr-30 versus Asp-30 and
Val-56 versus Ile-50 which increase the size and
hydrophobicity of the S
subsite of equine infectious anemia
virus PR(13) . The equine infectious anemia virus PR also shows
a stronger preference for hydrophobic residues at P
and
P
positions in the context of the VSQNYPIVQ sequence. In
MuLV PR, Val-39, Val-54, and Leu-92 act together in subsites S
and S
`. Unlike Val-39, which is located at the top of
the subsite, Val-54 and Leu-92 are at the sides of the subsites and are
less bulky than the equivalent Ile-47 and Ile-84, found in HIV-1 PR,
and therefore, they allow more bulky hydrophobic residues to fit well
at P
and P
` positions in MuLV PR.
Apart from
Val-54 and Ala-57, which contribute to S/S
`
subsites in MuLV PR, the mutation of other residues which are predicted
to form the flap region (e.g. Trp-53, Gln-55, or Gly-60) led
to a significant decrease of the PR activity. These results are in
agreement with other reports showing that mutations at the flap regions
of HIV-1 and Rous sarcoma virus PRs alter the catalytic rate of the
enzyme(24, 37) . Residues 47-56 at the top of
the flap region of HIV-1 PR are highly conserved among clinical
isolates(38, 39) , and their substitution by
site-directed mutagenesis often leads to the inactivation of the PR (37) . In some cases, viral resistance is associated with flap
mutations involving the replacement of Met-46 by Ile, Leu, or Phe (28, 29, 30, 34, 40) or
Gly-48 by Val(29, 31) . Multiple substitutions at the
flap region (e.g. M46I/I47V/I50V) have also been associated
with resistance to PR inhibitors(30) .
Although the subsites
of the substrate binding pocket of the retroviral PR are capable of
acting independently in the substrate selection, it will probably be
necessary to combine a set of mutations to fully alter the specificity
at any subsite. Interactions among different substrate-binding residues
seem to be important for substrate specificity. Amino acids forming the
S/S
` subsites and those involved in the flap
region appear to be critical determinants of specificity and catalytic
activity in MuLV PR, as well as in other retroviral PRs. A better
knowledge of the interactions between substrate and protease in various
retroviral PRs would be helpful to design broad spectrum inhibitors,
which would limit the emergence of drug resistant phenotypes in HIV.