Due to its crucial role in the early phase (1, 2, 3) as well as in the late phase of
viral life cycle (for review see (4) ), retroviral proteinase
(PR) (
)is a promising target for drug intervention; potent
inhibitors developed to block its action are now in advanced clinical
trials(5) . However, as in the case of reverse transcriptase
inhibitors, resistance rapidly develops, both in vitro and in vivo(6) . Studies to determine the common features
of the specificity of different retroviral PRs may help to design broad
spectrum inhibitors and reduce the possibility of viable mutants.
Therefore, we have extensively studied the specificity of HIV-1, (
)HIV-2(7, 8, 9) ,
EIAV(10, 11) , and bovine leukemia virus (12) PRs using oligopeptide substrates representing the
naturally occurring cleavage sites, as well as a peptide series
containing single amino acid substitutions in the P4-P3` (
)positions of the cleavage site sequence between the matrix
(MA) and capsid (CA) proteins of HIV-1. This MA/CA cleavage site
sequence has Tyr
Pro at the site of the cleavage (the down arrow
indicates the cleavable peptide bond). Recent analysis of retroviral
cleavage site sequences (13) and kinetic studies on HIV PRs (9, 14) suggest that two major types of cleavage sites
exist for retroviral proteinases: one having Tyr
Pro (type I) and
the other having mainly hydrophobic residues but not Pro (type II) at
the site of cleavage.
The avian type C viruses code the PR on the gag gene; therefore, unlike the other retroviruses, PR is made
in equivalent amounts to the structural proteins and was relatively
easy to purify. Many of the early studies on the role and specificity
of retroviral PR used AMV/RSV (
)as a model system (for
review see (15) ). RSV PR was the first retroviral enzyme for
which the crystal structure was determined(16) . Subsequently,
many crystal structures have been determined of HIV-1 and HIV-2 PRs in
the absence and the presence of inhibitors(17) . AMV/RSV PR is
still in focus of intensive specificity and mutagenesis
studies(18, 19, 20, 21, 22) .
Some RSV cleavage site peptides have been previously studied as
substrates of the retroviral enzymes(20, 23) . Here we
report the comparison of the AMV and HIV-1 proteinases based on a
complete set of substrates representing naturally occurring cleavage
sites. Recently Cameron et al.(21) used a series of
substrates containing single amino acid substitutions in the type II
NC/PR cleavage site sequence of RSV to compare the specificity of RSV
and HIV-1 PRs. Here, we compare the specificity of AMV and HIV-1 PRs
using a series of peptides containing single amino acid substitutions
in the type I MA/CA sequence of HIV-1. Detailed analysis of the results
by molecular modeling and comparison with previously published data on
retroviral proteinases has revealed the common characteristics of the
specificity of retroviral PRs as well as its strong dependence on the
sequence context of the substrate.
MATERIALS AND METHODS
Retroviral Proteinases
AMV PR purified from virus as described(24) , was
obtained from Molecular Genetic Resources (Tampa, FL). Recombinant
purified HIV-1 PR (25) used for the peptides representing
naturally occurring cleavage sites in RSV was a kind gift of Dr. Y. S.
E. Cheng (Experimental Station, The DuPont Merck Pharmaceutical
Company, Wilmington, DE). Active site titration of the enzymes was
performed using Pro-Pro-Cys-Val-PheSta-Ala-Met-Thr-Met for AMV PR (23) and a phosphinic acid-type substrate based inhibitor
(compound 3 in (26) ) for HIV-1 PR.
Oligopeptides
Oligopeptides were synthesized by standard tert-butoxycarbonyl or 9-fluorenylmethyloxycarbonyl chemistry
on a model 430A automated peptide synthesizer (Applied Biosystems,
Inc.) or a semiautomatic Vega peptide synthesizer (Vega-Fox
Biochemicals). Some peptides were synthesized by fragment condensation
as described previously(27) . Amino acid composition of the
peptides was determined with either a Durrum D-500 or a Waters
Pico-Tag amino acid analyzer. Stock solutions and dilutions were made
in distilled water (or in 10 mM dithiothreitol for peptides
containing Cys residues), and the peptide concentrations were
determined by amino acid analysis.
Enzyme Assay
The assays for the kinetic measurements were performed in
0.25 M phosphate buffer, pH 5.6, containing 5% glycerol (7.5%
glycerol for HIV-1 PR), 5 mM dithiothreitol, 1 mM EDTA, and 2 M NaCl as described previously (7, 8, 9) . The reaction mixture was
incubated at 37 °C for 1 h and was stopped by the addition of 9
volumes of 1% trifluoroacetic acid and then injected onto a Nova-Pak
C
reversed-phase chromatography column (3.9
150
mm, Waters Associates, Inc.) using an automatic injector. Substrates
and the cleavage products were separated using acetonitrile gradient
(0-100%) in water in the presence of 0.05% trifluoroacetic acid.
The cleavage of peptides was monitored at 206 nm, and the peak areas
were integrated. Amino acid analysis and/or N-terminal sequencing of
the collected peaks was used to confirm the cleavage sites in the
substrates. N-terminal sequencing was performed using a KNAUER Model
910 protein sequencer. The substrate concentrations used for the
kinetic measurements were in the range of 0.01-3 mM,
depending on the approximate K
values.
Kinetic parameters were determined at less than 20% substrate turnover
by fitting the data to the Michaelis-Menten equation using the
Gauss-Newton method using Fig.P program (Fig.P Software Corp., Durham,
NC). The standard errors of the kinetic parameters were below 20%.
Substrate hydrolysis followed Michaelis-Menten kinetics in the
concentration range of substrates used. For the determination of
relative activities, the reaction mixture containing 0.4 mM peptide was incubated at 37 °C for 1 or 24 h as described
previously(11, 12) . Amino acid analysis of the
collected peaks was used to confirm the site of cleavage with HIV
PR(9, 12) . For the AMV PR, cleavage products were
identified by the retention time, which was found to be identical to
that obtained with HIV PR. Relative activities were calculated from the
molar amounts of peptides cleaved per unit time by dividing the
activity on a given peptide by the activity on the unmodified substrate
SP-211 (Val-Ser-Gln-Asn-Tyr
Pro-Ile-Val-Gln), at less than 20%
substrate turnover, as described in Bláha et
al.(12) . Measurements were performed in duplicate, and
the average values were calculated. The error was less than 20%. The
relative activities for the HIV-1 PR have been reported
previously(11) .
Methods for Modeling Calculations
Initial Model
The crystal structures of the
native RSV PR with a modeled flap and peptide substrate (20) and HIV-1 PR complexed with a modeled substrate
Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln (9) were superimposed using
C
atoms and examined on a Silicon Graphics 310 computer graphics
system runnung the program CHAIN (28) or a Silicon Graphics
Indigo computer graphics system using the program Sybyl (Tripos Inc.,
St. Louis, MO). The residues forming the subsites for
Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln were determined previously for HIV-1
and HIV-2 PR(8, 9) , and the corresponding residues in
AMV/RSV PR were obtained from the structural alignment. Models of the
series of peptide variations of Pro-Ala-Val-Ser-Leu-Ala-Met-Thr that
represent the NC/PR cleavage site of RSV are described separately. (
)The proteinase substrate complexes were modeled with a
proton positioned midway between the closest side chain oxygens of the
two adjacent catalytic aspartates, Asp-25 and Asp-25`, and the carbonyl
oxygen of the scissile bond. The position of this proton has been shown
to be energetically stable by molecular dynamics
simulations(29) . All the crystallographic water molecules were
included because several appeared to be structurally important.
Energy Minimization Calculation
The minimization
and molecular dynamics were run using the program AMMP(30) . No
screening dielectric term or bulk solvent correction was included. A
constant dielectric of one was used. No cut-off was applied for
nonbonded and electrostatic terms, as described in Harrison and
Weber(29) . A modified version of the UFF potential set (31) was used as described separately.
These
modifications do not significantly change the performance of the
potential set on small molecules but result in constantly smaller root
mean square deviations between minimized and observed protein
structures. The atomic charges from the AMBER all-atom set were used
for the protein and water(32) . Parameters for water and for
the proton were as described(29) . The atomic positions for the
protein and water molecules were initially tethered to those in the
crystal structure of AMV PR in order to calculate and minimize the
hydrogen atom positions. The side chain atoms of the substrate peptide
were removed down to the C
atom for the substituted amino acids,
and the new atomic positions were created by a variation on distance
geometry(33) . The new atoms were minimized with respect to
bond, angle, torsion, and hybrid potentials. The proteinase structure
with nonhydrogen atoms from the crystal structure and minimized
hydrogen atoms was combined with each of the different peptides with
single amino acid substitutions. Then each of the side chain torsion
angles for substituted residues in the peptide substrate was rotated
through 360 ° in steps of 15 ° to search for alternate
conformations. This torsion search finds the angle(s) that have a
minimum in the nonbonded energy. The substrate atoms were minimized
using 300 steps of conjugate gradients. Finally, the complete model of
AMV PR with each different substrate was optimized by a longer
minimization using 100 steps of conjugate gradients followed by eight
cycles of alternating conjugate gradients (30 steps) and short runs of
molecular dynamics (20 fs steps at 300 K).
RESULTS AND DISCUSSION
Hydrolysis of Oligopeptides Representing Naturally
Occurring Cleavage Sites
Ten oligopeptides representing naturally occurring cleavage
sites in the RSV Gag and Gag-Pol polyproteins (Fig. 1) were
tested as substrates for AMV and HIV-1 PRs (Table 1). All
peptides were hydrolyzed at the expected site by the AMV enzyme, except
peptide 10, which was hydrolyzed at the Gly
Ile bond instead of
the Ala
Gly bond, the site in RSV IN reported by Grandgenett et al.(34) . For this peptide, the same site of
cleavage was also observed at low ionic strength, in the absence of
additional salt (data not shown). The RSV IN protein is phosphorylated
at the Ser residue near the cleavage site in the C terminus (P6
position of peptide 10 in Table 1; see (35) ). It should
be noted that phosphorylation was found to alter the C-terminal
processing of IN. It would be interesting to determine whether
phosphorylation of the serine would cause a shift in the cleavage site.
Kinetic parameters were determined at high (2 M) salt
concentration. High ionic strength was found to be optimal for AMV
PR(36) , similar to other retroviral
PRs(10, 37, 38) . The range of kinetic
parameters was compared with that we previously determined for the
lentiviral PRs using peptides representing cleavage sites in their Gag
and Gag-Pol polyproteins (Table 2). The K
was found to be also in the micromolar range, although one
peptide showed an exceptionally high value (peptide 9 in Table 1). However, the k
values were in a
10-30-fold lower range than those of other PRs (Table 2).
This is in good agreement with the relative amounts of the PR in the
virions. Although the PR of the HIVs and EIAV is coded for by the pol gene and is synthesized in an approximately
10-20-fold lower amount than the Gag proteins, the PR of AMV is
encoded in the gag gene (Fig. 1), and therefore it is
synthesized equimolarly to the structural Gag proteins.
Figure 1:
Cleavage
sites in RSV Gag and Gag-Pol polyproteins. Cleavage sites were
determined by sequencing purified virion proteins (reviewed in (15) ). The frameshift site is marked by an asterisk.
The peptides
were also assayed as substrates of HIV-1 PR. Only half of them were
substrates of this enzyme with similar kinetics to that obtained with
the AMV PR except for peptide 6, which was the best substrate of both
enzymes, with kinetic parameters comparable with the best values
obtained for peptides representing the HIV naturally occurring cleavage
sites (Table 2).
By comparing the amino acids at the P1-P1`
region of the cleavage sites, it is obvious that the three lentiviral
PRs prefer hydrophobic residues(7, 10) , whereas AMV
PR seems to tolerate glycine or polar residues. Nevertheless, the two
peptides with the best k
/K
values (peptides 4 and 6 in Table 1) contain hydrophobic
residues at both P1 and P1` positions.
Mapping of the Substrate Binding Site of AMV with a
Series of Peptides Containing Tyrosine and Proline at the Cleavage Site
Peptides containing single amino acid substitutions in the
sequence of the cleavage site found between MA and CA of HIV-1 were
assayed as substrates of AMV PR. Kinetic parameters for the unmodified
peptide (peptide 11 in Table 1) suggest that although it is a
poor substrate of AMV PR, it is comparable with some of the peptides
representing naturally occurring RSV cleavage sites. We have previously
determined relative activites compared with the unmodified peptide of
the HIV-1, HIV-2, and EIAV PRs for the substituted peptides to compare
the specificity of the enzymes(9, 11, 12) .
Relative activities of AMV PR are listed in Table 3. The k
/K
values determined for a
set of these substrates (data not shown) correlated well with the
relative activites (r = 0.987; n = 15).
Residues forming the substrate binding sites of HIV-1 have been
determined from the crystal structures of HIV-1 PR inhibitor complexes.
Based on homology (Fig. 2) and modeling of the substrate into
the RSV PR, residues predicted to form the respective subsites compared
with the HIV-1 PR are listed in Table 4together with the
respective HIV-1 residues and are shown in Fig. 3. By analogy to
the peptidic inhibitors(17) , the substrate is predicted to
bind in an extended
conformation, therefore alternative subsites
such as S3 and S1 are adjacent to one another (Fig. 3A). The results from the type I series of
peptides based on the HIV-1 MA/CA cleavage site can be compared with
those using type II peptides based on the RSV NC/PR cleavage site (21) in order to identify differences due to the sequence
context. Schematic representations of the HIV-1 MA/CA and RSV NC/PR
substrates together with the preferred substitutions are shown in Fig. 4.
Figure 2:
Sequence of HIV-1 and AMV proteinases. The
sequence of AMV PR differs only at two residues from RSV PR, the latter
contains Thr instead of Ala-52 and Leu instead of Val-82. These
residues are not expected to change the substrate
specificity.
Figure 3:
A, schematic representation of the HIV-1
MA/CA substrate in the S4-S3` subsites of PR. The relative size of each
subsite is indicated approximately by the area enclosed by the curved line around each substrate side chain. Proteinase
residues forming the subsites are shown for those that differ between
the AMV and HIV-1 PRs. The HIV-1 PR residues are in parentheses. Many of the residues contribute to more than one
subsite, as indicated by the position of the label. B,
stereoview of residues P4-P1 of the HIV-1 MA/CA cleavage site with the
substrate-binding residues that differ in AMV and HIV-1 PRs. The same
residues are shown as indicated in the scheme of A and Table 4. The AMV PR residues (thin continuous lines) are
from the crystal structure of RSV protease(16) . The HIV-1 PR
residues (dashed lines) and the substrate residues (thick
lines in a ball-and-stick representation) are from a
model based on the crystal structures of HIV protease with peptide-like
inhibitors. The modeling procedure is described under ``Materials
and Methods.''
Figure 4:
Schematic representation of the HIV-1
MA/CA (A) and RSV NC/PR (B) substrates. Preferred
substitutions for the HIV-1 peptide by the AMV PR (based on Table 3) and those for the RSV substrate by AMV PR (based on (21) ) are listed under the
residues.
S4 Subsite
The S4 subsite of AMV PR is near the surface
of the proteinase, as observed for other retroviral proteinases. In
this position the side chain of P4 residue may be partially exposed to
solvent. In accordance with this, polar substitutions (like Lys, Arg,
and Asn) of the original Ser in P4 resulted in fairly good substrates
of AMV PR (Table 3). However, compared with HIV proteinases,
substitution with bulky hydrophobic residues gave much higher activity
in our series of peptides (see Table 3). The best value was
obtained with the Ile substitution. AMV PR behaves similarly to EIAV PR
in preferring hydrophobic residues at P4(11) . However, with
the EIAV enzyme, the Leu substitution gave the best result, and the
increases in relative activities were much less pronounced. Compared
with HIV proteinases, EIAV PR contains extra residues leading to the
tip of the flap that could be involved in S4-P4 interaction, and the S4
pocket is predicted to be more hydrophobic than that of HIV
PRs(11) . Extra residues leading to the flap are also predicted
for AMV PR; modeling of RSV PR predicted that Pro-62 and Gln-63,
derived from the flaps, may interact with the side chain of
P4(20) . These additional amino acid residues in AMV/RSV PR are
predicted to provide a more enclosed S4 subsite, capable of
accommodating hydrophobic residues. When residues 61-63 were
deleted from AMV/RSV PR, the avian enzyme utilized substrates with
polar residues (Asn or His) in P4, similarly to HIV-1 PR(22) .
Based on mapping RSV PR subsites with a series of peptides based on the
NC/PR cleavage site, the original P4 Pro and its His substituted analog
gave the best result (Fig. 4), whereas substitution of Leu or
Phe resulted in nonhydrolyzable peptides(21) . This is
predicted to be due to the presence of
-branched Val in P2
position of the NC/PR peptide, which may restrict the internal space of
S4 for P4 residue, whereas in the MA/CA peptide the P2 Asn may allow
larger hydrophobic residues to be accommodated (Fig. 5).
Interestingly, MuLV PR also behaved similarly to EIAV PR and AMV PR but
differently from HIV PRs when mapped by the same Tyr
Pro peptide
series(38) . However, MuLV PR does not seem to have extra
residues leading to the flap but was predicted to have a more
hydrophobic pocket due to extra residues from another part of the
molecule.
Figure 5:
Interactions of P4 and P2 residues.
Residues forming the S4 to S2 subsites of RSV (thin continuous
lines) and HIV-1 (dashed lines) proteinases are shown
together with P3-P1 residues of two different substrates (in a ball-and-stick representation). The MA/CA substrate of HIV-1
with Leu at P4 is shown in thicker lines, and the NC/PR
substrate of RSV with Leu at P4 is in thinner lines. The HIV-1
PR residues and the substrate residues are from models based on the
cystal structures of HIV-1 PR with peptide-like
inhibitors.
Subsites S3 and S3`
With the P3 substitutions,
less dramatic results were obtained than with the P4 changes, as was
also found with EIAV and MuLV proteinases(11, 38) .
The ability of the S3 subsite to accommodate a variety of residues is
also seen with substitutions in the P3 position of the NC/PR cleavage
site peptide(21) . Substitution of Gln to Leu and especially to
Phe in the Tyr
Pro peptide provided substantially better
substrates ( Table 3and Fig. 4). Interestingly, the change
of P3` Val to Phe gave a dramatic increase in the relative activity,
whereas Leu did not provide any increase over the original peptide. The
S3 subsites of HIV-1 and AMV/RSV PR consist of relatively open and deep
pockets that are near the surface(21) . This could allow side
chains of amino acids in the P3 position freedom of movement to either
interact with hydrophobic residues near the S1 subsite or polar
residues at the surface of the enzyme(21) . In our series,
hydrophobic side chains are greatly preferred over polar residues. In
the NC/PR peptide, the P1 residue is a small Ser, whereas in our series
the large P1 Tyr may restrict the conformations available for the P3
residue. However, there may be a favorable hydrophobic interaction of
Phe P3 with the P1 Tyr side chain. Interestingly, Val and Leu
substitutions gave very poor substrates for EIAV PR (11) but
fairly good ones for AMV PR.The most marked changes in the S3
pocket of AMV PR are Arg-105` instead of Pro-81` and Gly-106` instead
of Val-82`. In both of these positions EIAV PR contains the same
residue as the HIV-1 PR. The preference for Phe in P3 by AMV/RSV PR
relative to HIV-1 PR can be explained by the presence of Gly-106` in
AMV/RSV PR, as compared with Val-82` in the analogous position of HIV-1
PR (21) and Val-87` of EIAV PR. Val-82` is at the top of the
HIV-1 S3 subsite pointing into the pocket (Fig. 3B) so
that it could sterically interfere with the binding of a large amino
acid residue such as Phe. This steric hindrance is not present with Gly
in the AMV/RSV subsite.
The AMV/RSV PR S3 subsite also contains 2
basic residues, His-65 and Arg-105, not found in the HIV-1 S3 subsite.
These residues confer on portions of the avian subsite a greater degree
of hydrophilicity(21) . However, the charged groups of these
amino acids are not positioned to provide strong ionic interactions
with substrate as evident from the very poor catalytic efficiency of
Asp substituted peptide. The aliphatic side chain of Arg-105 in AMV/RSV
PR may have an important hydrophobic role in defining the S3 subsite.
In agreement with these results, Konvalinka et al.(39) found that HIV-1 PR will accept a variety of residues
in the P3 position, whereas Strop et al.(23) reported
that the AMV enzyme has a preference for large polar or nonpolar
residues at this position.
Regarding the S3` pocket, the best value
was obtained with Phe substitution, similar to the results obtained
with the S3 substituted peptides. Substitution with Arg also gave a
substrate better than the original one. The preference for Phe over the
unsubstituted Val was also found for HIV-1 and HIV-2 PRs (9) and EIAV PR(11) . An interesting difference from
these enzymes is that they also prefer Leu in this position while the
AMV PR does not.
Subsites S2 and S2`
By changing the P2 Asn of
SP-211 to small or medium sized hydrophobic residues, a substantial
increase in relative activities was obtained, whereas for HIV-1 PR (and
also for HIV-2 PR), the original Asn gave the best results. Strop et al.(23) , using AMV PR, also found a preference for
small hydrophobic residues in S2. EIAV PR (11) and MuLV PR (38) also preferred medium sized hydrophobic residues at this
position, although the increases in relative activities or k
/K
values were much less
pronounced (maximum 10-fold increase) than observed for AMV PR. There
is a difference in the preferred size of the hydrophobic side chains.
For AMV and EIAV PR the smaller Ala, Cys, and Val gave the best results (Table 3., (11) ), whereas MuLV PR preferred the bulkier
aliphatic Leu and Ile(38) . However, it seems to be a common
result that Phe at P2 forms a poor substrate; modeling suggests that
the Phe side chain is too bulky for this subsite. Therefore, in the
type I sequence context, the preference for Asn by the HIV PRs is
rather exceptional, because the other studied retroviral proteinases
showed much higher preference for small or medium sized hydrophobic
residues.The S2 subsites of all PRs are sterically more restricted
compared with the S4 and S3 subsites and are predicted to accommodate
hydrophobic residues. The P2 side chain is surrounded by five Ile side
chains in AMV PR (residues 42, 44, 64, 108, and 67`), and three of them
are conserved in HIV-1 PR, but the residue corresponding Ile-44 is
Val-32, and that corresponding to Ile-42 is Asp-30. The ability of
HIV-1 PR to accommodate more polar residues may be related to the
presence of Asp-30(21) . In analogous positions, the Thr in
EIAV (11) and His in MuLV (38) also may be responsible
for accommodation of more polar P2 residues. The two residues that seem
to be crucial in determining the preference for Val over Leu at P2 in
our substrate series are Ile-44 and Ile-64. The corresponding side
chains of retroviral proteinases are shown in Table 5. Based on
this comparison, in both positions Ile favors Val at P2 in the
substrate, whereas Val favors Leu. However, in this respect the residue
equivalent to Ile-64 seems to be more crucial in the comparison of
HIV-1 and HIV-2 values. A Val preference over Leu was obtained for wild
type AMV/RSV PR, but a Leu over Val preference for HIV-1 PR using a
substrate series based on the NC/PR cleavage site and predicted to be
due to the role of Ile-44 compared with Val-32 in HIV-1
PR(21) . A mutant RSV PR containing Val in place of this Ile
resulted in higher relative activity than the wild type enzyme for the
Leu substituted peptide, but the preference for Val still
remained(22) . The removal of a methyl group in the side chain
of the substrate can be compensated for by the addition of a methyl
group to the side chain of the amino acid in the enzyme subsite, as
suggested by Cameron et al.(21) ; optimization of van
der Waals' interactions in the individual enzyme subsites is a
primary determinant in selection of amino acids in the different
substrate positions.
The S2` subsite, like S2, is also predicted to
accommodate smaller hydrophobic residues. In accordance with this, the
best result was obtained with Ala substitution. It is interesting to
note that the changes in relative activities are not as marked than as
for the P2 substitutions, the Ile residue of the unmodified peptide
could already provide good interactions. This also seems to be common
with EIAV PR and MuLV PR using the same substrate series (11, 38) and for HIV-1 and RSV PR using the NC/PR
based series (21) in which Ala at P2` gave the best
result(21) . For AMV PR, Strop et al.(23) reported an apparently symmetrical requirement for
small hydrophobic residues in both P2 and P2` and large hydrophobic
residues in P1 and P1`. However, their series was based on a type II
cleavage site containing two hydrophobic residues at both P1 and P1`
positions (Tyr and p-nitophenyl residues, respectively),
whereas our series was based on a type I (Tyr
Pro) cleavage site
peptide. Studying HIV proteinase specificity, marked differences were
found in subsite specificity of these two types of aromatic-Pro and
hydrophobic-hydrophobic (not Pro) cleavage sites; for example
-branched P2 residues are the best in type II sites, but they are
not optimal in type 1 sites due to steric collision with P1`
Pro(9, 14) .
Subsites S1 and S1`
Relative activities obtained
with the avian enzyme for the P1 substituted peptides were very similar
to those obtained with HIV-1 PR (see Table 3) and EIAV
PR(11) . None of the P1 modified peptides showed a relative
activity higher than that obtained with the unsubstituted peptide
except that containing Phe instead of Tyr. Also, the k
/K
values for HIV-2 PR (10) and MuLV PR (38) suggested a very similar
preference for the P1 side chains, having Phe > Tyr > Leu >
Met for AMV, HIV-1, EIAV and MuLV PRs (for HIV-2, Met substitution
produced a higher k
/K
value
than substitution with Leu). It is common in all cases that
substitutions having Gly, Ser, Asp, or Lys at P1 site gave
nonhydrolyzable substrates, whereas peptides with Ala, Val, Ile, or Trp
gave either noncleavable or very poor substrates. However, in the NC/PR
series Trp and Leu substitutions of the P1 Ser gave the best values.
The S1 and S3 subsites are overlapping (Fig. 6). The P3 Gln of
the MA/CA peptide restricts the available space for P1 residue, whereas
the P3 Ala in the NC/PR peptide does not (Fig. 6). In naturally
occurring cleavage sites for AMV, HIV-1, HIV-2, and EIAV and also of
other retroviral proteinases, hydrophobic amino acids predominate in P1
position(15) . Our results imply that the S1 binding site of
all the studied retroviral proteinases is very similar for the SP-211
substituted peptides and in this sequence context the S1 sites do not
contribute substantially to the observed differences in specificity of
the PRs.
Figure 6:
Interactions of P1 and P3 residues.
Residues forming the S1 and S3 subsites of RSV (thin continuous
lines) and HIV-1 (dashed lines) proteinases are shown
together with P3-P1 residues of two different substrates (in a ball-and-stick representation). The MA/CA substrate of HIV-1
is shown in thicker lines, and the NC/PR substrate of RSV with
His at P3 is in thinner lines. The HIV-1 PR residues and the
substrate residues are from models based on the cystal structures of
HIV-1 PR with peptide-like inhibitors.
Based on molecular modeling, the S1 subsite is mainly
hydrophobic, located deep inside the protein. Many of the residues
forming the S1 site of the HIV-1 and RSV PRs are conserved. However,
there are some nonconserved changes, including Val-104` (Thr-80`),
Arg-105` (Pro-81`), and Gly-106` (Val-82`). Arg-105` seems to
contribute to both S1 and S3 subsites, whereas Gly-106` renders the RSV
PR S1 pocket bigger than that of HIV-1 PR (Fig. 6). It is an
interesting feature of our substrate series that the P1 Tyr side chain
is predicted to occupy that region, which is also a part of the S3
pocket. The S1 and S3 subsites seem to be overlapping much more than
S4-S2, S2-S1`, S1-S2`, and S1`-S3` pockets. This could be a major
factor in sequence dependence of the results of specificity studies
based on different starting peptide sequences. Modeling also suggests
that depending on the P3 residue, OH of Tyr and the bulky Trp side
chain does not fit well into the AMV pocket, as was also observed for
HIV proteinases(9) .
It is worth noting that the peptide
containing Val at P1 was hydrolyzed by AMV PR, but that containing Ile
was not hydrolyzable. The peptides containing
-branched residues
(Val and Ile) at P1 of the MA/CA peptide were not hydrolyzable by HIV-1
and HIV-2 proteinases (9) and by EIAV PR(11) . Similar
results were obtained for HIV-1 PR using another peptide
series(40, 41) . However, a small rate of hydrolysis
was obtained with P1 Val and P1 Ile substituted SP-211 analogs with
MuLV PR, and this was attributed to the presence of Ala-57 instead of
Ile in HIV PRs. In the analogous position, AMV PR also contains Ile-57,
but the change of Gly-106 in place of Val-82 of HIV-1 may also provide
a somewhat larger substrate binding pocket, accepting
-branched
residue.
Another common characteristic for the studied retroviral
proteinases is that changing the P1` proline in the type I MA/CA
peptide to any other tested amino acids (see Table 3) gave
nonhydrolyzable or very poor substrates(9, 11) ,
whereas substitutions in Type II sequences provided good
substrates(21, 41) . These results indicate that
specificity at P1` position of the retroviral PRs is also strongly
dependent on the surrounding sequence.
Conclusion
Comparison of the specificity of the AMV PR to the HIV-1,
HIV-2, and EIAV proteinases using the type I MA/CA substrate series, as
well as comparing the AMV PR to HIV-1 PR specificity with the type II
NC/PR substrate series suggests that these PRs have many common
features. All prefer hydrophobic residues at the P1 position, although
the optimal size of the residue may depend on the residues forming S1
subsite and may also be a function of the residue at P3. P1` Pro is
unique in the type I MA/CA peptide, because changing it to any other
tested amino acid prevented hydrolysis by the retroviral proteinases ( Table 3and Refs. 9, 11, and 38). The size of the S2 and S2`
subsites is restricted, and these are predicted to be the smallest
subsites in all cases, but the preference for P2 is highly variable due
to the different PR residues forming the S2 subsite and also depends on
the P1` residue(9, 14) , P1, and perhaps the P4
residue. In the type I sequence context, the preference for Asn by the
HIV PRs is rather exceptional, because all of the other studied
retroviral proteinases showed much higher preference for small or
medium sized hydrophobic residues. Subsite S3 is more open than S2 and
can accept a variety of residues. Specificity in S3 is a function of
the P1 residue; a large P1 side chain restricts the size of the P3
residue that can be accommodated. Although S4 is close to the surface,
it shows a preference for hydrophobic residues except for HIV PRs,
although the size of the preferred residue is a function of P2. Our
results suggest that the specificity of retroviral proteinases is very
complex and strongly depends on the context of the substrate sequence.
The preference at a given position may depend not only on the
complementarity of residues at the same side of the
sheet (like
P3 and P1, P2 and P1`) but also on those at the opposite side. However,
modeling in many cases could give an explanation for the sequence
context dependence and is a promising tool to ``decode'' the
specificity of the retroviral proteinases. The strong sequence context
dependence should be taken into account in the design of proteinase
inhibitors, because developing resistance is one of the most serious
problems in treatment of AIDS. A mutation in a substrate binding
subsite of the PR indirectly could influence the specificity of the
other binding sites. Conversely, changing the ligand at those other
affected positions could complement the changes and regain the high
potency of the enzyme-ligand interaction.