(Received for publication, June 22, 1995)
From the
Analysis of mutational effects in the human immunodeficiency
virus type-1 (HIV-1) provirus has revealed that as few as four amino
acid side-chain substitutions in the HIV-1 protease
(M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant
to a panel of protease inhibitors either in or being considered for
clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M.,
Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins,
H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H.,
Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of
the molecular mechanism of drug resistance in AIDS therapies, the
three-dimensional structure of the HIV-1 protease mutant containing the
four substitutions has been determined to 2.4-Å resolution with
an R factor of 17.1%. The structure of its complex with MK639,
a protease inhibitor of the hydroxyaminopentane amide class of
peptidomimetics currently in Phase III clinical trials, has been
resolved at 2.0 Å with an R factor of 17.0%. These
structures are compared with those of the wild-type enzyme and its
complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P.
L., Culberson, C., Shafer, J., and Kuo, L. C.(1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration
of the protease due to the site-specific mutations. The C tracings of the two native structures are identical with a
root-mean-square deviation of 0.5 Å, and the four substituted
side chains are clearly revealed in the electron density map. In the
MK639-bound form, the V82T substitution introduces an unfavorable
hydrophilic moiety for binding in the active site and the I84V
substitution creates a cavity (unoccupied by water) that should lead to
a decrease in van der Waals contacts with the inhibitor. These changes
are consistent with the observed 70-fold increase in the K
value (
2.5 kcal/mol) for MK639 as
a result of the mutations in the HIV-1 protease. The role of the M46I
and L63P substitutions in drug resistance is not obvious from the
crystallographic data, but they induce conformational perturbations
(0.9-1.1 Å) in the flap domain of the native enzyme and may
affect the stability and/or activity of the enzyme unrelated directly
to binding.
The emergence of drug resistance remains a major bottleneck in
the pursuit of a long lasting, antiviral treatment against
AIDS(1) . When faced with selective pressure of an inhibitor,
some 20 of the 99 amino acid residues of the HIV protease undergo
mutations(2, 3, 4) . Sequence analyses of
virus isolates from patients participating in clinical trials have
revealed that various amino-acid substitutions in the HIV ()protease, in combination with as many as 10 or more
residues, are associated with the decrease in antiviral efficacy of the
protease inhibitor MK639 upon its prolonged usage in the course of 1
year (2) (Fig. Z1).
Figure Z1: Structure 1MK639.
MK639 (previously L-735,524) is
an inhibitor of peptide cleavages catalyzed by HIV-1 and HIV-2
proteases with Kvalues of 0.38 and 2.48
nM, respectively. It is effective against HIV replication in
cell culture and is efficacious in reducing viral load in carriers of
HIV-1(5) . Analyses of mutations in the HIV provirus have
revealed (2) that as few as four amino acid substitutions in
the HIV-1 protease (M46I/L63P/V82T/I84V) (
)suffice to yield
cross-resistance to a panel of protease inhibitors either in or being
considered for clinical trials, including MK639. To aid in the design
of inhibitors able to evade viral resistance, it is important to
understand the structural basis of resistance. We have previously
determined (6) the x-ray crystallographic structures of HIV-1
and HIV-2 proteases complexed to MK639 at 2.0- and 1.9-Å
resolution, respectively. Although four active-site residues (V32I,
I47V, L76M, V82I) are altered between the two enzymes, there are only
subtle changes in the binding mode of the inhibitor, suggesting that
the structural basis of resistance may not be discernible until the
degree of resistance becomes greater.
In this work, the native and
bound structures of a mutant HIV-1 protease, with a 70-fold change in
affinity (K = 26 nM)
toward MK639, have been determined. The observed structural features of
the MK639-bound mutant and wild-type proteases are consistent with the
extent of resistance raised against MK639.
The pET-3b-HIVPR plasmid containing the synthetic gene for
HIV-1 protease in the high expression vector pET3b was provided by Dr.
Jordan Tang, Oklahoma Medical Research Foundation(7) . A 354
base pair cassette was designed based on the wild type sequence of
pET-3b-HIVPR with appropriate nucleotide changes at positions 171 (G
C), 221 (T
C), 277 (T
A), 278 (T
C), 283 (A
G), and 285 (C
T) within the 333-base pair gene to yield
the quadruple (4X) mutant protease (M46I/L63P/V82T/I84V). The 4X
cassette was subcloned into the unique restriction sites Bpu1102I and BstEII within pET-3b-HIVPR to produce
pET-3b-HIV1PR-4X. Sequence-verified clones were transformed into
competent Escherichia coli BL21(DE3)pLysS (Ido and Tang cured)
host cells(8) . Cells were cultured, induced, and lysed as
described(7) . Enzyme purification, activity and inhibition
assays were conducted as described previously (7, 9) .
Crystallization of the mutant protease was accomplished at 4 °C
with use of the vapor diffusion method. The enzyme was prepared at 15
mg/ml in a pH 5 solution containing 10 mM MES, 1 mM EDTA, 1 mM DTT. The reservoir solution contained 0.6 M NaCl, 1 mM DTT, 3 mM NaN, and
0.1 M imidazole at pH 7. Hanging drops were prepared by mixing
the protein and reservoir solutions in 1:1 (v/v) ratio. Tetragonal
bipyrimidal crystals (0.9 mm
0.4 mm
0.4 mm) of the
mutant protease were obtained in 5-10 days. The cell constants
were a = b = 49.98 Å, c = 108.1 Å with 1 dimer/asymmetric unit. A 2.4-Å
resolution diffraction data set was collected on a RAXIS II imaging
plate using CuK
irradiation (Rigagu RU200 rotating
anode) generated at 50 kV and 100 mA. The data set, showing clearly
that the space group was P4
and not P4
22,
encompassed 29,001 measurements with 8,651 unique reflections (82.3%
completeness) and an R
([(
(I -
I
)
I]) of
3.3%. The coordinates (10) of the wild-type HIV-1 protease
were used as the molecular model. The initial R factor,
defined as
(
F
- F
)
F
, was 0.308. Using
the X-PLOR program package(11) , rigid body refinements of the
two monomers alternatively of the protease reduced the R value
to 0.239 using the data in the 8-Å to 3.5-Å resolution
range. Another cycle of X-PLOR refinement further reduced the R value to 0.226 at 2.4-Å resolution. A 2F
- F
map was calculated,
and a mutant protease model was built by employing the program CHAIN on
a Silicon Graphics system. The model was further refined; the final
model included 82 solvent molecules with an R factor of 0.171
for a total of 8,101 reflections. The R
was 0.312, and the r.m.s. deviations, from ideal values of
bond and angles, were 0.016 Å and 1.9°, respectively.
Crystals of the 4X protease complexed with MK639 were obtained at
ambient temperature in hanging drops under vapor diffusion conditions
against a solution of 0.1 M NaAc, pH 5.4, 0.5 M NaCl,
1 mM DTT, and 3 mM NaN. The cell
constants of the crystals were a = 60.06 Å, b = 86.90 Å, and c = 46.68 Å in
the space group P2
2
2. There was one protease
dimer per asymmetric unit. The diffraction data, collected as described
for the native enzyme and extending to 2.0-Å resolution, included
41,085 measurements with 13,590 independent reflections (86%
completeness) and an R
of 5.02%. The structure
of the inhibitor-bound HIV-1 mutant protease was determined with the
difference Fourier method employing the structure of the HIV-1 protease
MK639 as the model. The final model included 109 solvent molecules and
an R factor of 0.170 for reflections in the 6-Å to
2.0-Å resolution. The R
was 0.255.
The r.m.s. deviations of atoms of the structure from ideal values of
bond length and angle were 0.015 Å and 1.8°, respectively.
The inhibitor MK639 was found to bind the mutant HIV-1 protease in a
single orientation.
Fig. 1depicts in stereoview the C tracings of the wild-type HIV-1 protease and its quadruple (4X)
mutant. The r.m.s. deviation for all 198 C
atoms
between the 4X and the wild-type protease is 0.5 Å. (
)The largest deviations in the C
position
of these two molecules occur at the tip and in the hinge of the flap
loops. (
)The substituted side chains, Ile-46, Pro-63,
Thr-82, and Val-84, are highlighted for both subunits of the enzyme as
dictated by the 2F
- F
electron density map. Residues 82 and
84 are located in the active site of the enzyme. Residue 46 is in the
flexible flap loop of the enzyme, while residue 63 is near the hinge
region of the flap; both side chains point toward the bulk solvent.
Figure 1:
Stereo
tracings of the C backbones of the wild-type HIV-1
protease (green) and its quadruple mutant (pink) as
determined by x-ray diffraction data at 2.4-Å resolution. The
mutated side chains M46I, L63P, V82T, and I84V are shown in yellow for both subunits of the enzyme. Only residues 82 and
84 of both subunits are directly involved in inhibitor
binding.
Unlike the wild-type HIV-1 proteases, the two subunits of the 4X
mutant are not related by a 2-fold crystallographic axis in the
asymmetric unit. Nonetheless, the r.m.s. deviation for the C atoms of the two subunits is relatively small (0.37 Å). The
greatest difference between the subunits lies mainly in the flap loop
and in the peptide chains containing residues 15-20 and
64-72. (
)By excluding residues 15-20,
34-40, 48-52 and 64-72, the r.m.s. deviation for the
C
atoms in the two subunits is further reduced to 0.25
Å. For the four substituted residues between the subunits, the
displacements of the C
atoms are 0.2 Å for
Met-46, 0.31 Å for Leu-63, 0.2 Å for Val-82, and 0.18
Å for Ile-84.
Fig. 2illustrates the electron density of the quadruple mutant HIV-1 protease surrounding residues 46 and 63 as defined by x-ray diffraction. It reveals clearly that residues 46 and 63 of the mutant enzyme are isoleucine and proline as dictated by its DNA sequence. Similar unambiguous electron density definitions are found for V82T and I84V (electron density not shown).
Figure 2:
The
2F - F
electron density map, contoured at 1
level, of the
quadruple mutant HIV-1 protease surrounding (A) residue 46 and (B) residue 63. The refined model of the mutant enzyme is
shown in green and that of the wild-type HIV-1 protease is in red. The maps show that residues 46 and 63 of the mutant
enzyme are, respectively, Ile and Pro as predicted by DNA sequencing of
the mutant plasmid.
Fig. 3shows a view down the two-fold axis of the active site
of HIV-1 protease complexed with MK639. The r.m.s. deviation for all
C atoms between the 4X and the wild-type protease in
the bound mode is now only 0.2 Å. For clarity, the flaps of the
enzyme are only partially included in this figure. Residues Val-82 and
Ile-84 are highlighted in thickpinklines.
The Ile-84 and Ile-84` are symmetry-related by a two-fold rotation but
Val-82 and Val-82` are only pseudo symmetrically related because their
propyl side chains are oriented differently about the
C
-C
bond. The dashedlines mark the distances, within van der Waals radii, between atoms of
the inhibitor and the CD1 atoms of Ile-84/Ile-84`, as well as the CG2
and CG1 atoms of Val-82 and Val-82`, respectively. Related by a pseudo
two-fold rotation, the t-butyl and indanyl groups of MK639 are
bound in the S2 and S2` pockets of the enzyme. Correspondingly, the
pyridyl methyl piperidine and benzyl rings of the inhibitor are
situated in the S3/S1 and S1` pockets. A bound water molecule is seen
to be cushioned tetrahedrally between the tips of the flap loops
(Ile-50 and Ile-50`) and the 2 amide oxygen atoms of the inhibitor. (A
second bridging water molecule is found between the N2 amide nitrogen
of the inhibitor and the carboxyl oxygen of Asp-29`.) Not seen in Fig. 3are interactions of the hydroxyethylene group of the
inhibitor hydrogen bonded to Asp-25 and Asp-25` (beneath the inhibitor
structure). Altogether, seven atoms of MK639 are within hydrogen
bonding distances from atoms of the enzyme, either directly or
indirectly. These interactions have been reported in detail (6) for the HIV-2 protease. For comparison, the conformation of
MK639 (as seen in the active sites of HIV-1 and HIV-2 protease) are
shown in Fig. 4A.
Figure 3:
Stereo diagrams of the active site of
HIV-1 protease complexed with MK639 (previously L-725,524) as defined
by x-ray diffraction data at 2.0-Å resolution. The inhibitor is
depicted in green and the protease in thinpurple wire. Residues of Ile-50, Ile-50`, Val-82, Val-82`, Ile-84, and
Ile-84` are highlighted in thickpinklines.
The dashlines mark the distances within van der
Waals contacts between atoms of the inhibitor and the CD1 atoms of
Ile-84/Ile-84`, the CG1 atom of Val-82`, and the CG2 atom of Val-82.
Stick colored in yellow represents the positions of the side
chains of Thr-82, Thr-82`, Val-84, and Val-84` of the mutant protease
in the MK639 complex as defined by its electron density seen at
2.0-Å resolution and by super-posing the C backbone of the MK639-bound mutant enzyme onto that of the wild
type HIV-1 protease. The wild-type CG1 atom of Val-82` is now the
mutant OG1 atom of Thr-82` introducing an unfavorable interaction with
the inhibitor. The mutant OG1 atom of Thr-82 points away from the plane
of the stereoview and is shielded in this diagram; but its CG2 atom is
still within van der Waals distance (3.58-3.72 Å) from the
phenyl group of MK639 as opposed to a distance of 3.54-3.70
Å seen for the corresponding wild-type
atom.
Figure 4:
Comparison of the binding conformation of
MK639 as seen in the active sites of (A) the HIV-1 (green) and HIV-2 (yellow) proteases (see (6) for details) and (B) the HIV-1 protease (green) and the 4X mutant (purple) as defined by
x-ray diffraction data at 2-Å resolution. The inhibitors were
superimposed with the matrices derived by the least square fitting of
all C atoms of each pair of the proteases. For clarity,
the proteins are omitted in the
stereoviews.
In Fig. 3are also shown thicklines colored in yellow representing
the positions of the side chains of Thr-82, Thr-82`, Val-84, and
Val-84` in the MK639-complexed 4X mutant protease, as dictated by its
electron density and by super-positioning its C backbone onto that of the MK639-complexed wild-type HIV-1
protease. Both mutations in the active site introduce small alterations
(
0.3 Å) in their side chain positions. Most importantly, the
V82T substitution, while isosteric, introduces an unfavorable hydroxyl
moiety (OG1) at what was previously (wild type) the CG1 position of
Val-82` in the S1 pocket, within van der Waals distance (3.39 Å)
to the pyridyl methyl piperidine group of MK639. The corresponding
substitution (OG1 of V82T) in the S1` site points away from the
inhibitor and thus does not directly impact on binding because its CG2
atom is still within van der Waals distance (3.58-3.72 Å)
from the phenyl moiety. The I84V and I84V` substitutions, on the other
hand, symmetrically create in both the S1 and S1` pockets a small void
in bulk (
25 Å
) that can be expected to lead to a
decrease in van der Waals interactions with the piperidine group and
with the benzyl moiety of the inhibitor, respectively. (
)In
an apparently unsuccessful attempt to fill this void, the CD1 atom of
Ile-50` from the tip of the flap domain relocates by 1.29 Å
toward this pocket by rotating -35° about the
C
-C
bond and 170° about the
C
-C
bond; however, a similar change is
not seen for Ile-50. The structures of the bound MK639 in the active
sites of the wild-type and 4X mutant proteases are shown in Fig. 4B, revealing that the binding mode of MK639 is
essentially unchanged in the active site of the two enzymes. Together,
these results suggest that resistance against inhibitor binding, in the
case of MK639, is caused by subtle changes of the substituted side
chains (the introduction of an isosteric but unfavorable hydrophilic
group and the introduction of a smaller side chain to decrease van der
Waals contacts) rather than repositioning the bound inhibitor (due to
the introduction of a spatially hindering, larger side chain). The
side-chain positions of Thr-82 and Val-84 are also not different in the
open and closed forms of the mutant protease (data not shown),
indicating that these residues are not perturbed, within a r.m.s.
deviation of
0.2 Å, upon binding of MK639. The observed
changes in binding interactions of MK639 in the active sites of the 4X
protease are consistent with an attenuated affinity of this inhibitor
by a factor 70 (
2.5 kcal/mol), (
)a value sufficient to
render a potent protease inhibitor ineffective as an antiviral agent
against the HIV(2) .
Whereas structural contributions of
V82T and I84V toward resistance against MK639 are accountable with the
diffraction data shown here, the role of the M46I and L63P
substitutions is not obvious. These mutations away from the immediate
vicinity of the active site induce significant changes in the
C positions of residues in the flap domain of the
enzyme (see text above related to Fig. 1and Footnote 4), but
the significance of these changes is unclear. However, preliminary
kinetic results from our laboratory suggest that the combination of
M46I and L63P mutations affords the protease greater catalytic
efficiency than the native enzyme, and the V82T/I84V double mutations
render the HIV-1 protease a very poor enzyme. (
)Thus, it may
be that the M46I and L63P modifications, by introducing fine
adjustments to the protease conformation, compensate dynamically for
deleterious effects of the mutations (V82T and I84V) in the active
site.
Our observations are in contrast to those reported by Baldwin et al.(15) . These authors have observed
rearrangements of the HIV-1 protease C backbone around
residues 81-84 by up to 0.6 Å on binding of a symmetrical
inhibitor, A-77003, when Val-82 is substituted by an alanine. These
changes lead to a repacking of enzyme and inhibitor atoms in the S1 but
not S1` subsite in a manner that would diminish the potential loss of
binding affinity. The remainder of the mutant protease complex is
little altered from that of the wild-type enzyme (supporting our
interpretation that the changes seen in the flap domain of the 4X
mutant are due to the M46I and L63P substitutions). It is difficult to
generalize at this time the effect of resistance mutations of the
protease based on limited available data thus far. To further extend
our understanding of the structural basis of drug resistance in the
HIV-1 protease, we are pursuing determination of additional
inhibitor-bound structures of the 4X HIV-1 protease.