From the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India
Received for publication, June 28, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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The active site cleft of the HIV-1 protease (PR)
is bound by two identical conformationally mobile loops known as flaps,
which are important for substrate binding and catalysis. The present article reports, for the first time, an HIV-1 PR inhibitor,
ATBI, from an extremophilic Bacillus sp. The
inhibitor is found to be a hydrophilic peptide with Mr of 1147, and an amino acid sequence of
Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu. Sequence homology exhibited no similarity with the reported peptidic inhibitors of HIV-1
PR. Investigation of the kinetics of the enzyme-inhibitor interactions
revealed that ATBI is a noncompetitive and tight binding inhibitor with
the IC50 and Ki values 18.0 and 17.8 nM, respectively. The binding of the inhibitor with the
enzyme and the subsequent induction of the localized conformational
changes in the flap region of the HIV-1 PR were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-42 residues, which are present at the proximity of the flaps. We have demonstrated by fluorescence and circular dichroism studies that ATBI binds in the
active site of the HIV-1 PR and thereby leads to the inactivation of
the enzyme. Based on our results, we propose that the inactivation is
due to the reorganization of the flaps impairing its flexibility leading toward inaccessibility of the substrate to the active site of
the enzyme.
HIV-11 protease (PR) has
been classified as an aspartic protease that functions as a homodimer,
based on its primary amino acid sequence, its inhibition by pepstatin,
and its crystal structure (1-3). The retroviral protease is encoded in
the viral pro gene for all retroviruses, including HIV-1 (4,
5). During the replication cycle of HIV, gag and
gag-pol gene products are translated as polyproteins. These
proteins are subsequently processed by the virally encoded protease to
yield structural proteins of the virus core, together with essential
viral enzymes including the protease itself. The active site of HIV-1
PR is composed of the carboxylate side chains of two Asp residues, one
from each subunit of the dimer (6-10). The aspartic proteases are a
large family of enzymes with diverse functional roles that also share a
number of structural features (11-12). One such feature is called the flap, which lies above the active site cleft. By sequence alignment, the conserved sequence domain of the flap region begins at position 47 of the HIV-1 PR sequence and extends through the Gly at position 52. These residues form a short stretch of HIV-1 PR has been an attractive target for the development of drugs
against AIDS (15). The rational design of HIV-1 PR inhibitors may be
considered under two broad categories based on (a) the substrate specificity and (b) the structural homology of
HIV-1 PR dimer (16). Plethoras of synthetic inhibitory compounds
targeting the active site of the HIV-1 PR have been reported (17, 18). However, a lacuna of literature on biomolecules from microorganisms still exists. The present study deals with the isolation of an inhibitor, ATBI, of HIV-1 PR from an extremophilic Bacillus
sp. and the evaluation of its kinetic parameters. Fluorescence
spectroscopic studies revealed that ATBI binds in the active site of
the HIV-1 PR and is the first report of a noncompetitive inhibitor from an extremophilic microorganism. It is well established that the Trp-42
is present adjacent to the flaps, and the flap regions of HIV-1 PR are
the only dynamically flexible portions of the enzyme (19). We have
investigated the conformational changes induced in the flap regions of
the HIV-1 PR by monitoring the intrinsic fluorescence of the Trp
residues and the effects on the secondary structure of the HIV-1 PR, by
circular dichroism studies, upon binding of ATBI. We have also compared
the results obtained with that of the substrate and active
site-directed inhibitors of the HIV-1 PR. These results demonstrated
that the enzyme inactivation is caused by the loss of the flexibility
of the flaps restricting the entry and exit of the polypeptide
substrate and products.
Bacterial Strains and Growth Conditions--
The extremophilic
Bacillus sp. was grown on a liquid medium containing
soyameal (2%) and other nutrients at 50 °C for 48 h as
described (20) (the medium was adjusted to pH 10 by the addition of
sterile 10% sodium carbonate). The Escherichia coli strain harboring the recombinant plasmid containing the HIV-1 PR gene was
grown in M9 medium supplemented with ampicillin (40 µg/ml), thiamine hydrochloride (25 µg/ml), and glucose as the carbon source at 30 °C.
Purification and Biochemical Characterization of
ATBI--
Extracellular culture filtrate (1000 ml) of the
extremophilic Bacillus sp. was treated with activated
charcoal (65 g) and incubated at 4 °C overnight. The colorless
filtrate was subjected to membrane filtration through amicon-UM10
(molecular weight cut-off 10,000) and subsequently through amicon-UM2
(molecular weight cut-off 2000). The resulting inhibitor sample was
concentrated by lyophilization (50 ml). The residual concentrate was
further purified by reverse phase high performance liquid
chromatography (rp-HPLC). The concentrated inhibitor sample (100 µl)
was loaded onto a prepacked UltroPac column (Lichrosorb RP-18, LKB),
which was preequilibrated with 10% acetonitrile (CH3CN)
and 0.1% trifluoroacetate. The fractions were eluted on a linear
gradient of 0-50% CH3CN with H2O containing
0.01% trifluoroacetate at a flow rate of 0.5 ml/min and monitored at a
wavelength of 210 nm. The eluate was evaporated and lyophilized. The
residual matter was dissolved in distilled H2O and assayed
for the anti-HIV-1 PR activity. The active fractions were
rechromatographed on rp-HPLC under similar experimental conditions as
described above. The active peak was finally purified by rp-HPLC using
the Lichrosorb RP18 column.
The amino acid sequence of the purified peptide was analyzed with a
protein sequencer (Applied Biosystems model 476A), and the sequence
homology was done manually after retrieving the peptide sequences from
the data bank. Molecular mass of the purified ATBI was determined on a
VG Biotek Platform-II quadrupole electrospray mass spectrometer using
CH3CN-H2O (1:1) as mobile phase. The
isoelectric point of the inhibitor was determined as described
(21).
Enzyme Purification, Assay, and Kinetic Analysis--
The
recombinant HIV-1 PR harbored in Escherichia coli was
expressed by temperature induction after the onset of the log phase of
bacterial growth and purified by ammonium sulfate precipitation, dialysis, and gel filtration chromatography as described (22). The
HIV-1 PR activity was assayed using the synthetic substrate Lys-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-amide (23-24).
The HIV-1 PR was incubated at 37 °C at different concentrations of the substrate in a reaction mixture containing 100 mM NaCl,
5 mM Fluorescence and Circular Dichroism Analysis--
Fluorescence
measurements were performed on a PerkinElmer Life Sciences LS50
luminescence spectrometer connected to a Julabo F20 water bath. Protein
fluorescence was excited at 295 nm, and the emission was recorded from
300 to 500 nm at 25 °C. The slit widths on both the excitation and
emission were set at 5 nm, and the spectra were obtained at 500 nm/min.
Fluorescence data were corrected by running control samples of buffer
and smoothened. For binding studies, the HIV-1 PR (25 µg/ml) was
dissolved in 50 mM sodium acetate buffer (pH 5.6)
containing 100 mM NaCl, 5 mM EDTA, 5 mM
CD spectra were recorded in a Jasco-J715 spectropolarimeter at ambient
temperature using a cell of 1-mm path length. Replicate scans were
obtained at 0.1-nm resolution, 0.1-nm bandwidth, and a scan speed of 50 nm/min. Spectra were averages of six scans with the base line
subtracted spanning from 280 to 200 nm in 0.1-nm increments. The CD
spectrum of the HIV-1 PR (25 µg/ml) was recorded in 50 mM
sodium phosphate buffer (pH 5.6) containing 100 mM NaCl, 5 mM EDTA, 5 mM Purification and Biochemical Characterization of ATBI--
The
extracellular culture filtrate of the extremophilic Bacillus
sp. was subjected to activated charcoal treatment and ultrafiltration to remove the high molecular weight impurities. The concentrated inhibitor sample was further purified by rp-HPLC. The anti-HIV-1 PR
activity was associated with the peak A (Fig.
1a), and other eluted peaks
showed no inhibitory activity. Homogeneity of the active fractions was
indicated by the single peak as analyzed on rp-HPLC (Fig.
1b). Further, the purified ATBI showed a single band on an
analytical isoelectric focusing gel unit with a pI of 10.0. The amino
acid sequence of the purified inhibitor determined by a protein
sequencer was Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu and was
distinctly different from the sequence of the other reported inhibitors
of HIV-1 PR (32-35). The predominance of the charged amino acid
residues in the inhibitor sequence indicated its hydrophilic nature.
The molecular mass of ATBI as determined from electrospray mass
spectrometry was 1147 Da (Fig.
2a).
Kinetics of Inactivation of the Recombinant HIV-1 PR by
ATBI--
ATBI was found to inhibit the purified recombinant HIV-1 PR
with an IC50 value (50% inhibitory concentration) of 18 nM (Fig. 3a). The
inhibition of the HIV-1 PR followed a sigmoidal pattern with increasing
concentrations of the inhibitor. However, the secondary plot (the slope
of inhibition graph versus inhibitor concentration) was not
linear, suggesting that the application of Michaelis-Menten inhibition
kinetics was not appropriate in this study. The inhibition constant
Ki, determined by the classical double reciprocal
plot and also by Dixon plot was 17.8 nM (Fig.
3b), which is almost equal to the IC50 value of the inhibitor. The Lineweaver-Burk's reciprocal plot (Fig.
3c) showed that ATBI was a noncompetitive inhibitor of the
HIV-1 PR. For the inhibition kinetic studies, the HIV-1 PR activity was monitored in the presence of various concentrations of inhibitor and
substrate as a function of time. A very rapid inhibition of the HIV-1
PR was observed, which necessitated measuring all of the kinetic
parameters at second order association conditions. The Fluoremetric Analysis of Enzyme Inhibitor Interactions--
The
localized conformational changes induced in the HIV-1 PR due to the
interaction with ATBI were investigated by fluorescence spectroscopic
studies. The sequence data indicated the presence of four Trp residues
A-6, A-42, B-6, and B-42, two on each monomer of the HIV-1 PR (19). The
visualization and accessibility calculations of these Trp residues
revealed that they are present on the surface of the enzyme and thus
are excellent probes to monitor the changes in the tertiary structure
due to ligand binding. Therefore, the conformational changes induced in
the HIV-1 PR upon binding of ATBI were monitored by exploiting the
intrinsic fluorescence by excitation of the Secondary Structural Analysis of Enzyme Substrate-Inhibitor
Complexes--
To evaluate the effects of the inhibitor on the
secondary structure of the enzyme, we have analyzed the CD spectra of
the HIV-1 PR-ATBI complex. The secondary structure contents of the HIV-1 PR as determined from the crystallographic data were 4.04% The present paper describes a spectrofluorometric approach toward
investigating the localized conformational changes induced in the HIV-1
PR upon binding of the noncompetitive inhibitor ATBI. We have shown by
analyzing the kinetic parameters of the interactions of the HIV-1 PR
and the inhibitor that Michaelis-Menten kinetics cannot be applied for
this inhibition study. The failure of substrate protection against
HIV-1 PR inhibition by ATBI and the nondissociative nature of the HIV-1
PR-ATBI complex with multiple dilutions and washings led us to apply
tight binding inhibition kinetics. The short time observed for the
inhibition mandated performance of the kinetics under second-order rate
conditions. Observed Deciphering the crystal structure of the HIV PR and its inhibitor
complexes has gained immense interest among the crystallographers in
the last decade. From the available crystallographic data, it is
deduced that binding of substrate or peptide-analogue inhibitors in the
substrate-binding site of HIV-1 PR induces conformational changes in
the flaps (3, 14, 38). The apparent function of these flaps is to force
the peptide substrate into a The tryptophanyl fluorescence appears to be uniquely sensitive to
shielding by a variety of ligands because of the propensity of the
excited indole nucleus to emit energy in the excited state. The Trp
residues (A-42 and B-42) of the HIV-1 PR are present next to the
Lys-43, the first residue of the flap region, which extends from Lys-43
to Arg-57 (Fig. 7) (19). There have been
reports of introducing a Trp residue, which would act as a highly
specific reporter to monitor the structural changes in the flap regions by substrate-inhibitor binding (14). However, the site-directed mutagenesis studies of the HIV-1 PR have revealed that the enzymatic activity is extremely sensitive to mutations in the flap regions (39).
The inhibitors that bind to the active site also bind to the inner face
of the flaps of the HIV-1 PR. The binding of the inhibitor-substrate
and the subsequent movement of the flaps may have influence on the
intrinsic fluorescence of the Trp-42 residues. Based on the above
assumption, we have exploited these two Trp residues of the HIV-1 PR to
investigate the localized conformational changes induced upon substrate
or inhibitor binding. Our foregoing results have suggested that ATBI
binds in the active site of the enzyme and is a unique example where
the conformational changes in the flaps were investigated by monitoring
the radiative decay of the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-sheet followed by a turn
that ends with the conserved Gly at position 52 (13). The crystallographic structures of HIV-1 PR-inhibitor complexes
demonstrated that the binding of a peptide analogue inhibitor or a
peptide substrate involves numerous hydrogen-bonding interactions with the highly mobile flaps (13). Movement of these flaps apparently accompanies the binding of the peptide analogue or substrate, which
binds in an extended
-sheet, such that hydrogen bonds are established between the complementary carbonyl oxygens and the amide
protons of the peptide within the flaps. The presumed function of the
flap-peptide interactions is to entrap and align the scissile peptide
sequence in the HIV-1 PR active site (14).
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-mercaptoethanol, 5 mM EDTA, and 50 mM sodium acetate buffer, pH 5.6. After 15 min, the
reaction was stopped by the addition of an equal volume of 5%
trichloroacetate and followed by a 30-min incubation at 28 °C. The
cleavage products were analyzed by rp-HPLC and by decrease in optical
absorbance at 300 nm. The inhibition constant Ki was
determined as described by Dixon (25) and by Lineweaver-Burk's
equation. In Dixon's method, proteolytic activity of the recombinant
HIV-1 PR was measured at two different concentrations of substrate as a
function of inhibitor concentration. The kinetic constants were
determined by incubating the HIV-1 PR in the absence and presence of
ATBI with increasing concentrations of the substrate for 15 min at
37 °C. The inhibition was analyzed by the double reciprocal plot.
The kinetics of the HIV-1 PR inhibition was analyzed by a model for
tight binding inhibition (26). Kinetic determinations of enzyme
interaction with the inhibitor in the absence of substrate were
determined at short intervals by assaying the residual protease
activity. In these experiments, the residual enzymatic activity was
measured after the HIV-1 PR and the inhibitor were mixed and the
samples were subsampled at increasing time intervals and assayed with
the substrate. In all of the experiments, the inhibition of the HIV-1
PR was too rapid to measure under first order conditions. Rates of the
HIV-1 PR were therefore determined in all cases by second order
association rate kinetics. The association rate constants were
calculated according to the integrated second order rate equation
(27),
where [E] is the enzyme concentration, [I]
is the inhibitor concentration, and [EI] is the
concentration of enzyme-inhibitor complex. The residual values (free
enzyme) at 60 s were subtracted from that of the total enzyme, and
this gives the concentration of the enzyme-inhibitor complex. The
dissociation rate constants (k' or
(Eq. 1)
) were determined from
the formula
=
[I] by plotting the slope of the rate of
inhibition (
) or association rate constant (k") in each
reaction versus time multiplied by the inhibitor concentration. The slopes of the values were fitted by linear regression. Substrate protection studies were carried out by incubating the HIV-1 PR with different concentrations of substrate and then assaying the proteolytic activity at increased concentration of the inhibitor.
-mercaptoethanol. Titration of the enzyme with ATBI
was performed by the addition of different concentrations of the
inhibitor to the enzyme solution. For each inhibitor concentration on
the titration curve, a new enzyme solution was used. All of the data on
the titration curve were corrected for dilutions. Further, the emission
spectra of the HIV-1 PR were recorded in the presence of the substrate
and the active site-based inhibitors N-acetyl-Leu-Val-Phe-Al
(where Al is aldehyde) and pepstatin at 25 °C. Accessibility
calculations and visualization of Trp residues were performed by
Insight-II (28) from the crystallographic structure of the HIV-1 PR as
described (19).2
-mercaptoethanol in the
absence/presence of substrate (40 µM) or ATBI (20 nM). Secondary structure content of the HIV-1 PR, the HIV-1
PR-substrate complex, and the HIV-1 PR-ATBI complex was calculated
using the algorithm of the K2d program (30, 31).
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DISCUSSION
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Fig. 1.
rp-HPLC purification of ATBI. Shown is
the elution profile of ATBI obtained by rp-HPLC on a Lichrosorb RP-18
column preequilibrated with 10% acetonitrile and 0.1%
trifluoroacetate. a, 100 µl of the lyophilized ATBI sample
was loaded on a linear gradient of 0-50% acetonitrile with water
containing 0.01% trifluoroacetate for 15 min at a flow rate of 0.5 ml/min and monitored at 210 nm. The fractions containing the peaks
A, B, and C having retention times of
2.553, 3.268, and 3.853 min, respectively, were collected manually and
assayed for the anti-HIV-1 PR activity. b, 10 µl of the
fractions containing the peak A (associated with the anti-HIV-1 PR
activity) was reloaded onto the rp-HPLC system under similar
experimental conditions. The peak detected showed a retention time of
2.560 min.
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Fig. 2.
Chemical properties of ATBI. a,
the purified ATBI was analyzed for the determination of the molecular
mass using an acetonitrile/water (1:1) system as the mobile phase on a
quadrupole electrospray mass spectrometer. b, schematic
representation of the chemical structure of ATBI.
-values
obtained for ATBI were reasonably constant (data not shown), and the
average calculated value of
in the presence of substrate is
8.25 ± 0.50 × 10
4
s
1. The association rate constants
in the
presence and absence of substrate were calculated from the plot of the
HIV-1 PR inhibition versus time. The values of
(data not
shown) were not affected by the presence of the substrate, indicating
that the presence of substrate had no implication on the interaction of
the inhibitor and enzyme. The reciprocal of the values of
were
plotted as a function of the substrate concentration (Fig.
4a). The plotted line did not fit a linear plot but was a good fit for a
rectangular hyperbola, revealing noncompetitive inhibition. The
mechanism of inhibition of the HIV-1 PR was further deciphered by the
plot of
versus substrate concentration (Fig.
4b). Noncompetitive inhibition was represented by the
straight line. This plot is analogues to the
diagnostic plot of slope versus substrate concentration (36).
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Fig. 3.
Binding of ATBI to the HIV-1 PR and
inhibition kinetics analyses. a, the proteolytic
activity of the purified HIV-1 PR was determined in the presence of
increasing concentrations of ATBI. The percentage inhibition of the
HIV-1 PR activity was calculated from the residual enzymatic activity.
The sigmoidal curve indicates the best fit for
the percentage inhibition data obtained, and the IC50 value
was calculated from the graph. b, enzymatic activity of the
HIV-1 PR (25 µg/ml) was estimated using the substrate
Lys-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-amide (40 µM ( ) or 80 µM (
)) at different
concentrations of ATBI. Reciprocals of the reaction velocity were
plotted versus the inhibitor concentration. The
straight lines indicated the best fit of the data
obtained. The inhibition constant Ki was calculated
from the point of the intersection of the plots. c, the
HIV-1 PR (25 µg/ml) was incubated, without (
) or with the
inhibitor at 10 nM (
) and 20 nM (
) and
assayed at increasing concentrations of the substrate. The reciprocals
of the rate of the substrate hydrolysis for each inhibitor
concentration were plotted against the reciprocals of the substrate
concentrations. Ki was determined from the formula
as per the noncompetitive type of inhibition.
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Fig. 4.
Determination of the binding constants of the
HIV-1 PR and ATBI interactions. a, the HIV-1 PR and ATBI
were mixed, and the samples were removed at different time intervals
were assayed to determine the residual activity of the enzyme. The rate
constants were determined by the second order association kinetics. The
hyperbola indicated the best fit of the data obtained by plotting the
reciprocal of the association rate constant ( ) versus the
substrate concentration. b, Henderson plot of the change in
the slope of
for the inhibition of the HIV-1 PR by ATBI, as a
function of substrate. The straight line
represents the best fit for the data generated for the values of
.
-
* transition in the
Trp residues. The fluorescence emission spectra of the HIV-1 PR
exhibited an emission maxima (
max) at ~342 nm as a
result of the radiative decay of the
-
* transition from the Trp
residues, confirming the hydrophilic nature of the Trp environment. The
titration of the native enzyme with increasing concentrations of ATBI
resulted in a concentration-dependent quenching of the
tryptophanyl fluorescence (Fig. 5).
However, the
max of the fluorescence profile indicated
no blue or red shift, revealing that the ligand binding caused
reduction in the intrinsic protein fluorescence. A progressive
quenching in the fluorescence of the HIV-1 PR at 342 nm was observed
concomitant to the binding of substrate
(Lys-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-amide). Further, to throw light upon the mechanism of inactivation of the HIV-1
PR by ATBI, we have analyzed the interaction of two representative
competitive inhibitors, N-acetyl-Leu-Val-Phe-Al (where Al is aldehyde) (37) and pepstatin (2) by steady-state intrinsic
fluorescence measurements. The binding of the competitive inhibitors
led to the decrease in the quantum yield of the tryptophanyl fluorescence as indicated by the quenching of the emission spectra of
the HIV-1 PR. The comparative analysis of the intensity changes in the
fluorescence spectra of the HIV-1 PR upon binding of the substrate or
the known active site-based inhibitors was found to be similar to that
of ATBI, suggesting that ATBI binds in the active site of the
enzyme.
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Fig. 5.
Fluorescence emission spectra of the purified
HIV-1 PR. The fluorescence intensity spectra of the HIV-1 PR as a
function of the inhibitor are shown. Fluorescence was excited at
295 nm, and emission was monitored from 300 to 400 nm. Titration of the
enzyme was performed by the addition of different concentrations of the
inhibitor to the enzyme solution. The HIV-1 PR (25 µg/ml) was
dissolved in 50 mM sodium acetate buffer (pH 5.6)
containing 100 mM NaCl, 5 mM EDTA, 5 mM -mercaptoethanol. The concentrations of ATBI were 0 nM (
), 10 nM (
), 15 nM (
),
20 nM (
), 25 nM (
), 40 nM
(
), 45 nM (
), and 50 nM (
).
-helix, 47.47%
-sheet, and 48.49% of aperiodic conformation (19).2 The estimated secondary structure contents from the
CD analysis were 5%
-helix, 48%
-sheet, and 47% aperiodic
structure, which are in total agreement with the crystallographic data.
The circular dichroism spectrum of the HIV-1 PR-ATBI complex showed a
pronounced shift in the negative band at 220 nm of the native enzyme to
225 nm (Fig. 6). This shift reveals a
subtle change in the secondary structure of the enzyme upon ligand
binding. To elucidate the changes in the secondary structure of the
enzyme-inhibitor complex, we have compared it with that of the HIV-1
PR-substrate complex. Interestingly, the HIV-1 PR-ATBI and HIV-1
PR-substrate complexes exhibited a similar pattern of negative
ellipticity in the far-UV region, suggesting that the inhibitor causes
similar structural changes and was distinctly different from
that of the unliganded enzyme.
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Fig. 6.
Effect of the secondary stucture of the HIV-1
PR upon binding of the substrate or ATBI. Far-UV circular
dichroism spectra of the unliganded HIV-1 PR and its complexes with the
substrate and ATBI are shown. The HIV-1 PR (25 µg/ml)
dissolved in the buffer (as described under "Experimental
Procedures") and the CD spectra were recorded in the absence ( ) or
in the presence of the substrate 40 µM (
) or ATBI 20 nM (
) from 280 to 200 nm at 25 °C. Each spectrum
represents the average of six scans.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and
values for ATBI were independent of
the substrate concentration and relatively constant, implying that the
binding of the inhibitor was not influenced by the binding of the
substrate. However, a typical rectangular hyperbola resulted in a
reciprocal plot of 1/
versus [S]. We have concluded, by
using a diagnostic plot of
versus [S], that the
inactivation of the HIV-1 PR by ATBI was noncompetitive.
-sheet in the active site and to
correctly position its scissile bond between the two catalytic aspartyl
residues. The flaps accomplish this by the establishment of a series of
hydrogen-bonding interactions between amide nitrogens and carbonoyl
oxygens of the peptide substrate. Our interpretation for the changes
observed in the secondary structure of the HIV-1 PR due to the binding
of the substrate to the active site can be correlated to the inward
movement of the flaps. It is significant to note that the secondary
structure of the HIV-1 PR undergoes similar pattern of changes
upon binding of the substrate or inhibitor. Thus, we have attributed
the observed secondary structure changes in the HIV-1 PR-ATBI complex
to the inward movement of the flaps of the HIV-1 PR. The noncompetitive
nature of the inhibitor may be addressed due to the better binding
affinity of the inhibitor to the active site than the substrate. This, however, does not exclude the possibility of the differential binding
pockets for the inhibitor and the substrate in the active site of the enzyme.
-
* transition from the Trp residues
without mutating any of the residues in the flaps. The fluorescence
quenching of the HIV-1 PR by ATBI revealed that the binding of the
inhibitor reduces the quantum yield of the Trp emission. These results
were further corroborated by the quenching studies of the HIV-1 PR in
the presence of the substrate and the known competitive inhibitors. The
inhibition of the HIV-1 PR by N-acetyl-Leu-Val-Phe-Al
(where Al is aldehyde) and pepstatin is well documented. The quenching of the tryptophanyl fluorescence upon binding of the substrate or the
active site inhibitors can be very well explained by the shielding
effect of the Trp residues due to the inward movement of the flaps. The
comparison of the emission spectra of the HIV-1 PR upon binding of ATBI
with that of the substrate or the active site inhibitors led us to
conclude that ATBI binds to the active site of the enzyme and induces
the inward movement of the flaps, thereby reducing the radiative
decay of the intrinsic Trp fluorescence. The
concentration-dependent quenching of Trp fluorescence
showed that
max did not undergo any red or blue shift,
wherein the quenching of fluorescence was considerably high. These
findings indicated that the polarity of the Trp environment was
negligibly altered after binding of the inhibitor, suggesting minimal
conformational changes in the tertiary structure of the HIV-1 PR.
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Fig. 7.
Schematic representation of the proposed
mechanism of inhibition of the HIV-1 PR by ATBI. Secondary
structure of the HIV-1 PR is shown by the stereoview
ribbon diagram. The Trp residues A-42 and B-42
are adjacent to the flap region, whereas the Trp residues A-6 and B-6
are far from the flap region. The HIV-1 PR and other retroviral
proteases have the structural feature called the "flap region"
(shown above the arrows), which is important for
the substrate binding and catalysis. The binding of the inhibitor (as
indicated by the solid block) in the active site
induces inward movement of the flaps (as indicated by the
arrows). Further, we propose that the noncompetitive nature
of ATBI, along with its multiple nonbonded interactions with the flaps,
is responsible for the loss of the dynamic flexibility of the flaps,
resulting in the inactivation of the HIV-1 PR. The structure of the
HIV-1 PR is as described in PDB ID.1AID.
The majority of the inhibitors of HIV-1 PR are hydrophobic in nature.
There is a scarcity of hydrophilic peptidic inhibitors of HIV-1 PR,
which is significant for the bioavailability of the drug. Inhibition of
the HIV-1 PR by the hydrophilic peptides derived from the transframe
region of gag-pol has been reported (40). The transframe
octapeptide Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe, the N terminus of the
transframe octapeptide, and its analogues are competitive inhibitors of
the mature HIV-1 PR. The tripeptides Glu-Asp-Leu and Glu-Asp-Phe
derived from the transframe octapeptide were found to be most potent.
The x-ray crystallographic studies showed the interactions of Glu at P2
and Leu at P1 of Glu-Asp-Leu with residues of the active site of the
HIV-1 PR. As a hydrophilic inhibitor containing residues Asp, Ala, and
Glu, ATBI may have a similar mode of interaction with the residues in
the active site of the HIV-1 PR to that of the transframe octapeptide,
but it is not feasible to understand the interactions at the atomic level at present. However, the crystal structure will aid in
understanding the mechanism of inactivation of the HIV-1 PR by ATBI.
With the existing experimental evidence, we visualize that the charged side chains of the amino acids, the amide nitrogens, and the carbonoyl oxygen groups of ATBI could form many intermolecular hydrogen bonds and
other weak interactions (van der Waals, ionic, etc.) with the -sheet
of the flaps and with the other residues present in or near the active
site. Further, we propose that the tight binding and noncompetitive
nature of ATBI in conjunction with the multiple nonbonded interactions
may be sufficient to cause the loss of the dynamic flexibility of the
flaps, which is crucial for the substrate binding and catalysis of the
enzyme. A schematic diagram representing the
proposed mechanism is depicted in Fig. 7. The noncompetitive nature of
the ATBI indicated that the inhibitor-complexed form of the HIV-1 PR
loses its binding ability to the substrate, since the flaps can no more
open up for the substrate to be aligned in the active site of the HIV-1
PR, which subsequently results in the inactivation of the enzyme. These
observations are at variance with the binding of the substrate to the
enzyme in the absence of ATBI, where the flexibility of the flaps can
be regained after catalysis.
Inhibitors directed toward the active site of the HIV-1 PR are well
documented (41, 42). Despite their loss in potency due to the
spontaneous mutations occurring in the active site (29, 43) leading
toward the drug resistance behavior of the virus, there is a paucity of
literature on noncompetitive inhibitors. A constant search for the new
class of HIV-1 PR inhibitors with high potency is a frontier area of
biomedical research. The side chains of the peptidic inhibitor ATBI,
which are capable of forming many nonbonded interactions (hydrogen
bonding, van der Waals interactions, etc.) with the enzyme, might
result in superior resistance characteristics in comparison with the
competitive inhibitors. ATBI, as proposed, could interact with the
backbone of the -sheet of the flaps of the HIV-1 PR, thus
eliminating the probability of drug resistance by a single mutation.
ATBI, by virtue of its unique sequence and noncompetitive mode of
inhibition, may represent a new class of inhibitors of the HIV-1 PR and
could open up a new horizon for the development of lead molecules.
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ACKNOWLEDGEMENTS |
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We thank Prof. J. Kay (Department of Biochemistry, University of Wales College of Cardiff, Wales, United Kingdom) and Drs. N. Roberts and A. D. Broadhurst (Department of Virology, Roche, Welwyn Garden City, UK) for the E. coli clone containing the recombinant HIV-1 PR gene; Dr. P. Ratnaswamy (National Chemical Laboratory, Pune, India), Dr. K. V. S. Rao (International Center for Genetic Engineering and Biotechnology, New Delhi, India), and Prof. A. Wlodawer (National Cancer Institute-Frederick Cancer Research and Development Center) for valuable discussions; U. Kale (Bioinformatics Center, University of Pune, Pune, India) for the visualization and secondary structure rendering; and Dr. K. N. Ganesh (Organic Chemistry Synthesis Division, National Chemical Laboratory, Pune, India) for permitting the use of the spectropolarimeter, fluorescence spectrophotometer, and SYBYL software.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Council of Scientific and Industrial Research,
Government of India.
§ To whom all correspondence should be addressed: Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India. Tel.: 91-20-589-3034; Fax: 91-20-588-4032; E-mail: malarao@dalton.ncl.res.in.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M005662200
2 Secondary structure as given by the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) of HIV-1 PR as in PDB ID.1AID.
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus type 1; PR, protease; rp-HPLC, reverse phase high performance liquid chromatography; Nle, norleucine.
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