(Received for publication, February 4, 1997, and in revised form, April 30, 1997)
From the Department of Biochemistry, University
Medical School of Debrecen, H-4012 Debrecen, Hungary, the
¶ Department of Pharmacology, Jefferson Cancer Institute, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107, the
Laboratory of Cellular and Developmental Biology, NIDDK,
National Institutes of Health, Bethesda, Maryland 20892, the ** Special
Program in Protein Chemistry and the
Molecular Virology and Carcinogenesis
Laboratory, Advanced BioScience Laboratories-Basic Research Program,
NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 21702-1201
Two major types of cleavage sites with different
sequence preferences have been proposed for the human immunodeficiency
virus type 1 (HIV-1) proteinase. To understand the nature of these
sequence preferences better, single and multiple amino acid
substitutions were introduced into a type 1 cleavage site peptide, thus
changing it to a naturally occurring type 2 cleavage site sequence. Our results indicated that the previous classification of the retroviral cleavage sites may not be generally valid and that the preference for a
residue at a particular position in the substrate depends strongly on
the neighboring residues, including both those at the same side and at
the opposite side of the peptide backbone of the substrate. Based on
these results, pseudosymmetric (palindromic) substrates were designed.
The retroviral proteinases are symmetrical dimers of two identical
subunits; however, the residues of naturally occurring cleavage sites
do not show symmetrical arrangements, and no obvious symmetrical
substrate preference has been observed for the specificity of HIV
proteinase. To examine the role of the asymmetry created by the peptide
bonds on the specificity of the respective primed and nonprimed halves
of the binding site, amino acid substitutions were introduced into a
palindromic sequence. In general, the results suggested that the
asymmetry does not result in substantial differences in specificity of
the S3 and S3 subsites, whereas its
effect is more pronounced for the S2 and S2
subsites. Although it was possible to design several good palindromic
substrates, asymmetrical arrangements may be preferred by the HIV
proteinase.
The specificity of retroviral proteinases has been studied
intensively using both polyproteins and oligopeptides as substrates (for review, see Refs. 1-3). These studies have provided a basis for
the rational design of potent, selective inhibitors. Various proteinase
inhibitors are now in clinical trials or approved for therapy (for
review, see Refs. 4-6). Comparison of cleavage site sequences of human
immunodeficiency virus type 1 (HIV-1)1 and
type 2 (HIV-2) suggested that the enzyme had a broad specificity and
lacked consensus substrate sequence (7). Initially three types of
cleavage sites were proposed for HIV-1, HIV-2, and simian immunodeficiency virus (8). Subsequently, two major types of cleavage
sites were proposed for retroviral proteinases, type 1 having
-Tyr(Phe)*Pro- and type 2 having hydrophobic residues (excluding Pro)
at the site of cleavage (9-11). These two types of cleavage sites were
proposed to have different preferences for the P2 and
P2 positions, where the peptide bond between
P1 and P1
is cleaved (notation is according to
Ref. 12). Our studies with type 1 substrates indicated a preference for
small residues like Cys or Asn at the P2 position and a
preference for
-branched Val or Ile at the P2
position
(10). The lower catalytic constants with P2
-branched
residues were predicted to be due to steric collision with
P1
Pro (10). On the other hand, using a series of peptides
based on a type 2 cleavage site,
-branched residues, especially Val,
were found to be favorable at P2, whereas Glu was preferred
at P2
(11). Interestingly, Griffith et al. (11) found Glu as the preferred P2
residue in a peptide series,
when the P2-P1
sequence of a type 1 cleavage
site (-Asn-Tyr*Pro-) was substituted into a type 2 substrate. Although
some of the differences of the subsite specificity in type 1 and type 2 cleavage sites were explained by molecular modeling, most of the
dependence of the specificity of proteinases on the sequence context is
unexplored.
The HIV-1 proteinase is a dimer of two identical subunits. It exhibits an exact crystallographic, 2-fold rotational (C2) symmetry in the structure without inhibitor (for review, see Ref. 4). Based on this symmetry, the potential advantages of C2 symmetric HIV-1 proteinase inhibitors including high selectivity, potency, and stability were proposed, and structurally symmetric HIV-1 proteinase inhibitors were designed containing two amino-terminal halves of a putative substrate (13). Crystal structures of HIV-1 proteinase with inhibitors can be either symmetric or asymmetric (4). The symmetry or asymmetry was initially thought to arise from the symmetry or asymmetry of the inhibitor, but even crystal structures of HIV proteinase with symmetric inhibitors can have asymmetric proteinase subunits (14).
Considering the symmetry of the HIV proteinase, a symmetrical
preference for substrate residues would be expected for naturally occurring cleavage sites, since the high mutation rate could readily evolve such sequences. However, there is no obvious preference for
symmetrical sequences in HIV proteinase cleavage sites (listed in Ref.
15) or in other retroviral proteinase cleavage sites (see Ref. 1).
Using a series of oligopeptides containing single amino acid
substitutions in a naturally occurring type 1 cleavage site peptide,
the respective P and P positions (for example, P2 and
P2
) appeared to be similar, but substantial differences were also found. For example, the peptide containing P2 Asn
was a very good substrate; however, Asn at P2
resulted in
a poor substrate (10). It was not clear whether these differences were because of the asymmetrical interactions of the peptide amides and
carbonyl oxygens in the substrate or intramolecular interactions of the
substrate side chains (10).
To explore further the dependence of HIV proteinase specificity on the sequence context of its substrates we introduced single or multiple substitutions into a type 1 cleavage site peptide and changed it to a naturally occurring type 2 cleavage site sequence. Based on the results obtained, we designed a pseudosymmetric (palindromic) substrate and introduced amino acid substitutions into this sequence to explore the effect of asymmetry created by the peptide backbone on the different specificities of the respective primed and nonprimed proteinase subsites. To study whether the enzyme prefers pseudosymmetric (palindromic) or asymmetric arrangements of the substrate residues, we have also studied the doubly substituted (also palindromic) versions of the starting pseudosymmetric substrate.
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). All peptides were synthesized with an amide end. 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 AssayPurified HIV-1 proteinase was prepared as described previously (16). Active site titration for the HIV-1 proteinase was performed with compound 3 (17). The proteinase assays were performed in 0.25 M potassium phosphate buffer, pH 5.6, containing 7.5% glycerol, 1 mM EDTA, 2.5 mM dithiothreitol, 0.1% Nonidet P-40, 2 M NaCl in the presence of 8-140 nM enzyme. The reaction mixture was incubated at 37 °C for 1 h, and the reaction was stopped by the addition of guanidine HCl (6 M final concentration). The solution was acidified by the addition of trifluoroacetic acid, and an aliquot was injected onto a Nova-Pak C18 reversed-phase chromatography column (3.9 × 150 mm, Waters Associates, Inc.) using an automatic injector. Substrates and the cleavage products were separated using an increasing water-acetonitrile gradient (0-100%) in the presence of 0.05% trifluoroacetic acid. Cleavage products of proteinase-catalyzed hydrolysis for these peptides were identified by amino acid analysis and/or by NH2-terminal sequencing. Kinetic parameters were determined by fitting the data obtained at less than 20% substrate hydrolysis to the Michaelis-Menten equation by using the Fig. P program (Fig. P Software Corp.). The substrate concentration was 0.01-5.0 mM depending on the approximate Km values.
Molecular Modeling and Energy MinimizationThe structures were examined on Silicon Graphics computers running the program Sybyl (Tripos Inc., St. Louis, MO) or CHAIN (18). The starting model for the HIV-1 proteinase with the substrate Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln (asterisk indicates the site of cleavage) was described previously (19). All the other enzyme-substrate structures were built from this model by altering the side chain(s) of the appropriate residue(s). Each of the side chain torsion angles for substituted residues in the peptide substrate was rotated through 360° in steps of 15° to find the conformation with the smallest nonbonded energy as described (20).
Energy minimization and molecular dynamics of the modified substrates were run using the program AMMP (21), as described previously (19). Finally, the model structures were examined in the computer graphics system.
Substitution of Amino Acids of a Type 1 Cleavage Site Peptide with Residues of a Type 2 Substrate
Previously, we performed extensive comparisons of the
specificities of HIV-1 and HIV-2 proteinases using oligopeptides
representing naturally occurring cleavage sites in their Gag and
Gag-Pol polyproteins (15). These cleavage sites have been classified as
type 1, which contains an aromatic amino acid and Pro at P1
and P1, respectively, and type 2, which has mainly
hydrophobic residues but not Pro at the site of cleavage. We showed
that an oligopeptide (peptide 1 in Table I) representing
the cleavage site in p66 of HIV-1 for generating the p51 subunit of the
heterodimeric reverse transcriptase of HIV-1 and another peptide
(peptide 2 in Table I) representing the homologous sequence in p68 of
HIV-2, and therefore proposed to be the cleavage site (22), were
substrates of the HIV proteinases (15). These peptides, which match
type 2 cleavage site sequences, were the starting points for our design
of palindromic substrates since they are partly symmetric with aromatic
amino acids at the P1, P1
, and they contain
negatively charged residues at the P3 and P3
positions. In addition, peptide 2 also contains Thr at both
P2 and P2
positions. Furthermore, peptides 1 and 2 with the exception of the P2
residues share the same
sequence in the P4-P3
region, which is the
major determinant for specificity (23). However, peptide 2 was found to
be a much poorer substrate of the HIV proteinases than peptide 1 (see
Table I and Ref. 15).
|
Subsequently Fan et al. (24) demonstrated that in fact the sequence of peptide 2 does not represent the actual cleavage site required to be cleaved to produce the smaller subunit of the HIV-2 reverse transcriptase. They found that the real cleavage site has the sequence of AFAM*ALTD and is downstream from the one proposed by Le Grice et al. (22). Nevertheless, we in this study and Fan et al. (24) have confirmed our initial finding that the HIV-2-derived peptide 2 or its shorter octapeptide homolog is a substrate of the HIV-1 proteinase.
We have also compared the specificity of the HIV-1 and HIV-2
proteinases using a series of oligopeptide substrates containing single
amino acid substitutions in the sequence of SP-211 (see peptide 3 in
Table I), a peptide that corresponds to the type 1 MA/CA cleavage site
in HIV-1 (10, 23). In these studies it was found that substitution of
Pro at the P1 position to any other amino acid tested,
including Tyr, formed nonhydrolyzable or very poor substrates of HIV
proteinases. These P1
-substituted peptides inhibited the
hydrolysis of SP-211 by HIV proteinase, which suggested that they were
able to bind to the enzyme (10). The best inhibition was obtained with
the P1
Tyr-substituted peptide,2 suggesting its high affinity for
the HIV-1 proteinase. In good agreement with these preliminary
findings, both the Km and
kcat values determined in the present study were
substantially lower for the P1
Tyr-substituted peptide
compared with the unmodified one (compare peptides 3 and 4 in Table I).
Substitution of P1
Pro of SP-211 with Tyr converts a type
1 substrate to a type 2 substrate. However, the P1
Tyr-substituted peptide is a poor substrate of HIV-1 proteinase
(peptide 4 in Table I). To understand better the specificity of HIV-1
proteinase and the differences of the subsite preference in type 1 and
type 2 cleavage sites, further single and multiple substitutions were
carried out in the type 1 MA/CA cleavage site substrate (peptide 3),
introducing residues characteristic of the type 2 cleavage site
peptides 1 and 2 (Table I). The same substitutions were also introduced in the P1
Tyr-modified (type 2) substrate. This peptide
series allowed us to compare the preference for P2 Asn over
Thr as well as P2
Ile over Thr in different sequence
contexts. The ln (kcat/Km) values are directly related to the free energy of the binding of the
transition state by the enzyme (25). If the subsite interactions are
mostly independent of each other, the
kcat/Km ratios (equal to
e
G/RT) for substrate pairs having
X and Y residues at the same subsite, should be
similar and independent of the surrounding sequence, as has been found
for e.g. trypsin (26) and chymotrypsin (27).
Single substitutions of P2 Asn and P2 Ile with
Thr (peptides 5 and 6 in Table I, respectively) resulted in substantial
increases in Km and decreases in catalytic constants
compared with the unmodified peptide 3. Comparison of kinetic
parameters of peptides 1 and 2 as well as of peptides 3 and 6 suggests
that
-branched hydrophobic residues such as Val or Ile at
P2
positions are much more favorable than the
-branched
but more hydrophilic Thr in these two different sequence contexts.
Furthermore, a similar preference was found in two other series of
peptides (11). Single substitutions of P3 Gln and
P3
Val to Asp (peptide 7 and 8 of Table I, respectively)
resulted in dramatic increases in Km values but only
moderate decreases in kcat values, as was found previously for cleavage with HIV-2 proteinase (10).
Substituting Thr at P2 residue for Asn in the
P1 Tyr analog of SP-211 (peptide 9 in Table I) did not
yield substantial changes in the kinetic parameters, whereas
substitution of P2
Thr for Ile in the same sequence
context (peptide 10) yielded an approximately 10-fold increase in
(kcat/Km). The same
substitution was very unfavorable in the P1
Pro-containing
peptides (compare peptides 3 and 6 in Table I), suggesting a strong
influence of the P1
residue on the preference for the
P2
residue. Substitution of Thr at both the P2
and P2
positions of peptide 4 was less effective than the
single P2
substitution (compare peptides 10 and 11 of
Table I). Interestingly, whereas the P3
Asp substitution of peptide 4 yielded a substrate that was even less susceptible to
hydrolysis (peptide 12 in Table I), a further substitution for
P2 Thr (peptide 13) yielded a substrate with
Km and kcat/Km values better than
those of the naturally occurring type 2 cleavage site peptide 1. As
expected from the comparison of peptides 1 and 2, further substitution
of P2
Ile for Thr yielded a substrate with much lower
kcat/Km value because of the
substantial increase of the Km (peptide 14 of Table I).
Enzyme-substrate models were built and energy minimized to explore the
possible interactions of the enzyme and the substrates at the molecular
level. The substrate lies in an extended -conformation in the
substrate binding site, which puts P4, P2,
P1
, and P3
residues at one side and
P3, P1, and P2
on the other side
(Fig. 1). Adjacent substrate binding sites (like
S2 and S1
) partially overlap (Fig. 1).
Molecular modeling suggested that the P2 Thr-substituted SP-211 analog could not fit well in the S2 subsite (not
shown) because of the predicted interaction with P1
Pro,
as described previously for another
-branched amino acid, Val (10).
When the P1
Pro is changed to Tyr, this restraint is
removed. However, comparison of peptides 4 and 9 suggested that this
substitution alone is not sufficient to make Thr preferable over Asn in
the SP-211 sequence context, but the additional P3
Val to
Asp exchange is required (peptides 12 and 13). However, a further
substitution of P2
Ile to Thr again resulted in Asn over
Thr preference at P2 (peptides 14 and 15, see Table
II).
|
It is of interest to note that substitution of P1 Pro to
Tyr in various sequence contexts yielded much lower
Km and kcat values
(e.g. compare peptides 3 and 4, 5 and 9 etc.). Both the
substrate backbone and the proteinase residues of the S1
(and S1) subsites may move to accommodate the large Tyr at P1
(and P1) by moving toward the overlapping
and adjacent subsites S2-S3
(S3-S2
) and therefore restrict the space
available for other subsites (see Fig. 1). Similar movements of a
mutated residue in the substrate binding site have been observed in the
crystal structure of an inhibitor-resistant proteinase (28). The
restriction of the available space at the neighboring subsites could be
a major determinant in the sequence context-dependent
preference for P2 and P2
. In the
-P2-Tyr*Tyr-P2
-Asp- context there is a preference for one larger and one smaller residue at P2 and
P2
suggesting complementarity of these sites. In the case
of Ile at P2
, the smaller Thr is preferred over Asn at
P2 (peptides 12/13 in Table II), whereas in case of Thr at
P2
the larger Asn is preferred over Thr at P2
(peptides 14/15 in Table II). Conversely, with the larger Asn at
P2 the smaller Thr is preferred at P2
(peptides 12/14), whereas with the smaller Thr at P2 the
larger Ile is preferred at P2
(peptides 13/15). In the
case of the -P2-Tyr*Tyr-P2
-Val- context the
preference is less distinct. In accordance with the suggested
P2-P2
complementarity, with P2
Thr there is a preference for the larger Asn at P2
(peptides 10/11 in Table II), whereas with Asn at P2 there
is a preference for the smaller Thr at P2
(peptides 4/10
in Table II). However, when P2 is Thr, P2
Thr was preferred over Ile (peptides 9/11 in Table II). Apparently the
hydrophobic,
-branched Val at P3
may restrict the type
of amino acid which can be accommodated optimally in the
S2
subsite.
In summary, results on the preference of Asn over Thr at P2
and Ile over Thr at P2 positions (Table II) suggest a very
strong dependence on sequence context. For example, P2
preference depends not only on the P1
, but also on the
P2 and P3
residues. Strop et al.
(29) suggested a context dependence in the
P2-P2
residues in the specificity of avian
myeloblastosis virus proteinase, which was also demonstrated by Ridky
et al. (30). Our results indicate that P3
, and
likely other outer distal residues like P4 and
P3, may also substantially influence the preference for a
subsite. The change of preference at the P2
position as a
function of P1
and P3
residues (Table II)
also suggests that the context dependence of the specificity of HIV-1
proteinase is not restricted only to residues located at the same side
of the peptide backbone. Molecular modeling and inspection of the
crystal structure of HIV proteinase-inhibitor complexes suggest that
the peptide backbone of the substrate does not occupy a rigid position,
and depending on the substitutions, it could move toward the S or S
sites. SP-211 contains the relatively small Pro at P1
,
which may allow a relatively larger S2
subsite, and this
peptide showed Ile over Thr preference (peptide 3/6 in Table II). A
large hydrophobic residue at P1
, such as Tyr, could force
the peptide backbone toward the S2
site, making it
smaller. This could be the reason for the Thr over Ile preference at
P2
in the case of the P1
Tyr-substituted
peptides (see peptides 4/10 in Table II).
Substitutions in a Palindromic Substrate Sequence
S3 and S3Peptide 16 (Table I) contains a palindromic sequence at the
P3-P3 positions.
Elimination of P5 Val and substitution of P4
Gln for Ser resulted in a completely palindromic substrate (peptide 17 in Table III). It is important to note that this shorter
peptide showed 10-fold higher kcat and
kcat/Km values than peptide
16. The effect of residues P5 and P4 on
substrate hydrolysis has been reported previously (11, 23). To compare
the specificity of the S3 and S3
substrate
binding pockets in an identical sequence context, the Asp residues of
the palindromic peptide 17 were substituted to Gly, Gln, Phe and Leu
residues (Table III). Although the Gly and Gln residues were also
doubly substituted, kinetic measurements were not possible for the
doubly substituted Phe and Leu peptides because of low solubility
(peptides 26 and 29 in Table III). Substitution of the P3
and P3
Asp to Gly (peptides 18 and 19, respectively) resulted in similar Km and
kcat values and only a 2-fold decrease in
kcat/Km values.
Interestingly, changing both P3 and P3
Asp
residues to Gly yielded a much higher Km value
(peptide 20). Single substitutions to Gln also resulted in moderate
changes in the kinetic parameters, whereas the double substitution
yielded a substantial reduction of the kcat
value. Whereas substitution to P3 Phe resulted in a 4-fold
decrease in Km and kcat
(peptide 24), the P3
Phe substitution did not cause such a
big effect (peptide 25). Substitution of P3 or P3
Asp to Leu resulted in a substantial decrease in
catalytic constants.
|
Modeling showed that the same residues of the enzyme interact with the
appropriate subsite at both sides of the scissile bond (Table
IV). S3 and S3 subsites are
near the surface, and they are formed by hydrophobic residues and two
charged residues (Table IV). In the starting palindromic substrate
(peptide 17) the P3 and P3
Asp residues can
interact with Arg-8
and Arg-8, respectively (Fig. 2)
and compensate for the lower van der Waals energy compared with Phe in
P3 and P3
positions. Surprisingly, the
catalytic constants of the Gly-substituted peptides are comparable to
those obtained with Phe and Asp substitutions. Apparently,
P3 and P3
in this sequence context do not have
a substantial effect on the efficiency of hydrolysis. The largest
differences between the P3 and P3
mutated
peptides were obtained with Leu substitutions. Also, these peptides had
the lowest kcat/Km values.
Previously, we had proposed that the interaction between the
hydrophobic P3
residue of SP-211 (see peptide 3 in Table
I) and Phe-53 of the enzyme may play a crucial role in proper closing
of the flexible flap (10). In a molecular dynamics simulation of the
flap movement, Phe-53 and its symmetry counterpart were among the few
residues suggested to be involved in the "triggering" event (31).
In the palindromic sequence context, although peptides with hydrophobic residues at P3
have lower Km values
than those with polar residues or Gly (Table III), the differences are
not as dramatic as in the case of SP-211, where at least 10-fold
differences were found (peptides 3 and 8 in Table I; Ref. 10). In our
palindromic sequence context, the effect of a hydrophobic residue at
P3
resembles that of P3 in SP-211 in which
only a moderate increase of Km was obtained by
introducing nonhydrophobic residues at P3 (10). A
P1
Tyr residue in the pseudosymmetric context may
partially occupy the overlapping S3
pocket as
P1 Tyr can partially occupy the S3 (Fig.
3), resulting in less dependence on P3
hydrophobicity for the proper closing of the flap.
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Only moderate differences were found in the kinetic parameters of
substrates having identical substitutions at the P3 and P3 sites, except for the Leu substitutions. These results
suggest that the asymmetry of the peptide backbone does not play a
major role in the large differences of the specificities of the
respective S3 and S3
subsites we had
previously established (10). Instead, the specificity differences are a
consequence of the different sequence context of the substrates.
S2 and S2 SubstitutionsThe P2
or P2
Thr residues of peptide 17 (Tables III and
V) were substituted with Gly, Glu, Cys, Ala, Ile, and
Leu residues (Table V). Because of the low solubility, the doubly
substituted Ile and Leu substrates were not assayed (peptides 44 and
47, respectively). Single substitutions of the Thr residues with Gly
resulted in substantial increases in Km values and
decreases of kcat and
kcat/Km values compared with
peptide 17, whereas the doubly substituted peptide was not hydrolyzed
(peptides 31-33 in Table V). Previous studies showed that Gly in these
positions does not form good substrates, independently of the sequence
context (10, 32). Substitution of P2 Thr to Glu resulted in
a large increase in Km value; however, this peptide
showed the highest kcat value among all of the
substituted peptides (peptide 33 in Table V). P2
Glu
substitution (peptide 34) resulted in a less dramatic increase in
Km and unchanged kcat values.
P2
Glu was found to be optimal in both type 1 and type 2 sequence context by Griffith et al. (11). This preference
was predicted to be because of hydrogen bonding of the side chain
carboxyl to the backbone NH of residues 29
and 30
of the enzyme (11).
Also, P2
Glu is frequently found in viral and cellular
protein cleavage sites of HIV-1 proteinase (33). At the
P2-P2
region the type 1 substrate used by
Griffith et al. (11) contained residues (-Asn-Tyr*Pro-Ile-)
identical to those in SP-211; however, P2
Glu substitution
was an unfavorable substitution in the SP-211,2 suggesting
the importance of residues outside the P2-P2
determining P2
preference, as we have also found with the
multisubstituted peptides (Table II). Therefore, preference for Glu at
P2
position of type 1 and type 2 cleavage sites is also
strongly dependent on the surrounding substrate residues. Single
substitutions of the palindromic peptide 17 with small hydrophobic
residues such as Cys (peptides 36-37) and Ala (peptides 39-40) at
P2 and P2
resulted in somewhat better
Km and kcat values, as well as higher kcat/Km values. It
is interesting to note that the double Cys substitution (peptide 38)
resulted in a substantially higher Km value and a
less efficient substrate than the single substituted ones. Also, the
double Ala substitution (peptide 41) did not improve the substrate over
the P2 Ala-substituted one, although P2
Ala
was also favorable over Thr in single substitution. These findings
suggest that symmetrical (palindromic) arrangements do not necessarily
give better substrates than asymmetrical arrangements. The
substitutions with Ile gave lower Km and increased catalytic constants (peptides 42-43). Only P2
Leu was a
preferable substitution, whereas P2 Leu substitution was
not (peptides 45 and 46, respectively).
|
The S2 and S2 subsites of HIV-1 proteinase are
also hydrophobic, but they are smaller than the S3 and
S3
sites. The P2 side chain is surrounded by
the hydrophobic Val-32, Ile-47, Ile-50, Leu-76, and Ile-84 residues.
The same amino acid residues of the other subunit form the
S2
. Comparing the kinetic parameters of the P2
and P2
substituted substrates, the small hydrophobic Ala was a better substitution at P2 than at P2
,
whereas the larger Cys, Ile, and Leu residues were more preferred at
P2
, suggesting that the S2
subsite may be
somewhat larger than the S2. Interestingly, the largest
differences in kcat/Km values
were found between peptides having Leu substitutions in P2
and P2
(compare peptides 45 and 46 in Table V) as well as
P3 and P3
(compare peptides 27 and 28 of Table
III). Because of the asymmetry of the peptide bonds, the pseudosymmetry
center of the substrates is shifted approximately by 0.5 Å,
positioning the carbonyl oxygen rather than the center of the scissile
bond into the line of the C2 symmetry axis of the
proteinase dimer. This deviation puts the P2
residue
farther from the center of the enzyme compared with P2
(Fig. 4), toward the open end of the binding site, which may be a reason why larger residues are better at P2
than
at P2. The effect of this deviation is stronger close to
the center, which may be the reason for larger differences obtained for
the P2 and P2
substitutions than for the
P3 and P3
substitutions. Furthermore, whereas
S3, S3
subsites are exposed at the surface, the S2, S2
subsites are more restricted
because they are smaller and inside the proteinase.
Hydrolysis of Substrates Having Palindromic Sequences
The hydrolysis of peptide 17 and its doubly substituted
derivatives in Tables III and V showed that peptides with completely palindromic sequences could be substrates of HIV-1 proteinase. Based on
mirroring the NH2-terminal side of the sequence of the naturally occurring MA/CA (VSQNY*PIVQ) and CA/NC cleavage site (TATIM*MQRGN), we synthesized other palindromic peptides, and these
were also found to be substrates of the enzyme (peptides 48 and 49 of
Table VI). Simultaneous substitution of P2
and P2 residues of peptide 49 to Ala (peptide 50) produced
little change in kcat/Km.
Since Tyr is better in P1 than Met in both type 1 (10) and
type 2 cleavage site sequences (2), Tyr was substituted for Met at both
P1 and P1
, which resulted in the most
efficient palindromic substrate in our series (peptide 51). These
results suggests that various palindromic substrates could be designed
for HIV-1 proteinase, and specificity studies using these substrates
could eliminate the differences caused by the sequence context on the
preference of the respective S and S
sites.
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It is also of interest to note that exchange of P1 and
P1 residues of SP-211 as well as mirroring the P
residues
resulted in nonhydrolyzable peptides (peptides 52 and 53 of Table VI). These results also indicate the nonequivalent nature of the respective S and S
subsites.
The dependence of the specificity of HIV-1 proteinase on the
sequence context of its substrate peptides was studied by making single
and multiple substitutions of amino acids in naturally occurring
cleavage site peptides, and palindromic substrates were designed.
Although it was possible to design palindromic substrates, our results
suggest that a symmetric arrangement of residues may not be favorable.
The S3-S3 region of the HIV proteinase is
generally hydrophobic. However, a completely hydrophobic sequence may
not provide soluble oligopeptides. Furthermore, the naturally occurring cleavage sites in retroviruses are expected to be in regions connecting folded protein domains of polyproteins, where a high degree of hydrophobicity may be unfavorable for the correct conformation. Besides
the hydrophobicity, the size of residues also appears to be important.
Our results suggest that in the case of tight packing of S1
and S1
with Tyr residues, a relatively larger residue at
either P2 or P2
is preferable, but not at both
positions. These factors may explain why symmetric subsite arrangements
have not evolved at the naturally occurring cleavage sites, despite the
symmetric nature of the retroviral proteinases.
Previous studies did not explain whether the rather large differences
in specificity of respective S and S subsites are attributed to the
asymmetry introduced by the binding of the substrate and/or the
different amino acid sequence context. Design of a palindromic substrate and substitutions in its respective positions allowed us to
study these effects. In general, our results suggest that the
previously established differences in specificity of P3 and P3
positions could be mainly due to the different sequence
context: in these positions the asymmetry introduced by the peptide
backbone of the substrate does not seem to play an important role. For P2 and P2
positions the effect of asymmetry is
not negligible; however, the different sequence context could be still
the predominant cause of the substantial differences found previously
(10).
Our results also show that the earlier classification of retroviral
cleavage sites into two types is an oversimplification. Preference of
Asn in P2 and Ile in P2 in type 1 cleavage
sites as well as Ile (Val) in P2 and Glu at P2
in type 2 cleavage site substrates seems to be a function of the
residues outside of the P2-P2
region of the
substrate. The enzyme-substrate interaction of the HIV-1 proteinase as
well as other retroviral proteinases cannot be described residue by
residue, but the neighboring residues of the substrate should also be
taken into account. These neighboring residues include not only the
residues at the same side of the peptide backbone but also residues at
the opposite side as suggested by Strop et al. (29) from
analysis of avian myeloblastosis virus proteinase specificity. Similar
results were obtained by Ridky et al. (30) from kinetic
analysis of substrates containing double substitutions from
P3 to P1
in the NC/proteinase cleavage site peptide of Rous sarcoma virus. However, the specificity pattern is even
more complex than was originally thought, not only the internal
P2-P2
residues, but outer residues like
P3
may also substantially influence the preference for a
subsite, even for positions separated by four residues in the peptide
sequence.
The most important features of the sequence context dependence are the
size and -branch in a residue. P1
Tyr, unlike Pro, may
fill completely the large S1
subsite and could restrict
the space for substrate residues at other subsites. On the other hand, substitution of Pro at P1
could eliminate unfavorable
interactions with
-branched residues at P2 (10, 30).
Even small changes in the substrate sequence might cause rearrangement
of the substrate residues, as seen in some enzyme-inhibitor complexes
as well as predicted by molecular modeling. This strong sequence
context dependence also raises difficulties for predicting cleavage
sites. For example, an early prediction method developed by Poorman
et al. (7) failed to predict the cleavage site in Nef
protein (34). New prediction methods that take into account the
context-dependent nature of the specificity (35) might give
better results. Also, molecular modeling and improved energy
calculation methods may help to develop better methods for
predictions.
We thank Tunde Csire for technical assistance; Cathy Hixson and Suzanne Specht for help with the amino acid analysis; Ivo Bláha, Eva Majerova, and Patrick Wesdock for peptide synthesis; David Cavanough for help in molecular modeling; Jin Wu for preparing the figures; and Carol Shawver for preparing the manuscript.