Báo cáo khoa học: Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site potx

Crystal structures of HIV protease V82A and L90M mutants reveal
changes in the indinavir-binding site
Bhuvaneshwari Mahalingam
1,
*, Yuan-Fang Wang
1
, Peter I. Boross
1,2
, Jozsef Tozser
2
, John M. Louis
3
,
Robert W. Harrison
1,4
and Irene T. Weber
1,5
1
Department of Biology, Georgia State University, Atlanta, GA, USA;
2
Biochemistry and Molecular Biology Department, University
of Debrecen, Hungary;
3
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases,
The National Institutes of Health, Bethesda, MD, USA;
4
Department of Computer Science, Georgia State University,
Atlanta, GA, USA,
5
Department of Chemistry, Georgia State University, Atlanta, GA, USA
The crystal structures of the wild-type HIV-1 protease (PR)

and the two resistant variants, PR
V82A
and PR
L90M
,have
been determined in complex with the antiviral drug, indi-
navir, to gain insight into the molecular basis of drug
resistance. V82A and L90M correspond to an active site
mutation and nonactive site mutation, respectively. The
inhibition (K
i
)ofPR
V82A
and PR
L90M
was 3.3- and 0.16-
fold, respectively, relative to the value for PR. They showed
only a modest decrease, of 10–15%, in their k
cat
/K
m
values
relative to PR. The crystal structures were refined to
resolutions of 1.25–1.4 A
˚
to reveal critical features associ-
ated with inhibitor resistance. PR
V82A
showed local changes
in residues 81–82 at the site of the mutation, while PR

L90M
showed local changes near Met90 and an additional inter-
action with indinavir. These structural differences concur
with the kinetic data.
Keywords: aspartic protease; crystal structure; drug resist-
ance; HIV-1.
Inhibitors of the HIV-1 protease are effective antiviral drugs
for the treatment of acquired immune-deficiency syndrome
(AIDS). However, their therapeutic efficacy is limited owing
to the rapid selection of drug-resistant mutants of the
protease. Analysis of clinical isolates has revealed extensive
mutations in 45 residues of the 99-residue protease that are
associated with resistance to protease inhibitors [1,2]. Indi-
navir was one of the first protease inhibitors used as an
antiviral agent to treat AIDS. Resistance to indinavir arises
by a combination of different mutations in the protease gene
[3,4]. A high level of resistance is associated with substitu-
tions of up to 11 residues in the protease, although different
combinations of these mutations have been observed [5].
Mutations of the conserved residues V82 and L90 are among
those most commonly observed in protease inhibitor treat-
ments [2], and are frequently observed, even in indinavir
monotherapy [4]. Drug-resistant mutants of HIV protease
are expected to show reduced sensitivity to specific inhibitors,
while maintaining sufficient enzymatic activity and specific-
ity for viral maturation and infectivity. However, single
protease mutations and specific combinations can have
lower viral infectivity than wild-type HIV. Drug-exposed
HIV with multiple protease mutations, including resistant
substitutions of M46I/G48V/L90M and F53L/A71V/V82A,

produce defects in polyprotein processing and reduced viral
infectivity [6]. Therefore, it is important to understand the
molecular basis for the altered activity and structural changes
of these resistant mutants as compared to the wild-type
protease, in order to understand the molecular mechanism
of resistance and to develop new antiviral therapies.
Crystal structures show that HIV protease forms a
binding site that consists of subsites S3–S4¢,whichspan
about seven residues (P3–P4¢) of a peptide substrate [7]. The
clinical inhibitors primarily bind in subsites S2–S2¢.Struc-
tural and kinetic studies of resistant protease mutants
have shown a range of effects that depend on the specific
combination of mutation with substrate or inhibitor, as
well as the assay conditions. Mutations observed in drug
resistance have been classified either as substitutions in the
active site (inhibitor-binding site) that directly influence
inhibitor binding, or as substitutions of nonactive site
residues with indirect influences. Previously, mutants with
either increased or decreased catalytic activity, inhibition
constants, and stability relative to the wild-type enzyme
were observed, independently of the location of the
mutation [8–14]. We have analyzed high-resolution crystal
structures of the mature HIV-1 protease bearing either
single or double substitution mutations bound to substrate
analogs [13,15]. Some of these mutants showed structural
changes consistent with differences in their enzymatic
activity. Crystal structures of an inactive mature protease
bearing the mutations D25N and V82A, in complex with
inhibitors ritonavir or saquinavir, and substrates, show
differences in the interactions of inhibitors as compared to

Correspondence to I. T. Weber, Department of Biology, Georgia State
University, PO Box 4010, Atlanta, GA 30302-4010, USA.
Fax: + 1 404 651 2509, Tel.: + 1 404 651 0098,
E-mail:
Abbreviations: PR, wild-type HIV-1 protease; RMS, root mean
square.
*Present address: Renal Unit, Massachusetts General Hospital,
Charlestown, MA 02129, USA.
(Received 19 December 2003, revised 19 February 2004,
accepted 27 February 2004)
Eur. J. Biochem. 271, 1516–1524 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04060.x
substrates [16]. The structural differences compared to the
wild-type enzyme help to explain the resistant phenotype.
Previously, the crystal structure of the HIV protease in
complex with indinavir was reported at a resolution of 2.0 A
˚
[17,18]. The crystal structures of several multiple mutant
drug-resistant HIV proteases with indinavir have also been
reported at 2–2.5 A
˚
resolution [18–20]. In these structures it
is difficult to interpret the effect of a single mutation. Hence,
we describe the crystal structures of an optimized wild-type
HIV-1 protease (termed PR, see the Experimental proce-
dures), and drug-resistant mutants, PR
V82A
and PR
L90M
,in
complex with indinavir refined at 1.30-, 1.4- and 1.25 A

˚
resolution, respectively. Atomic details from these very
high-resolution structures will be essential for the design
of second-generation inhibitors against HIV-1 protease, to
offset drug resistance.
Experimental procedures
Purification of HIV-1 protease constructs
The wild-type mature HIV-1 protease (PR) [21], optimized
for structural and kinetic studies, bears five mutations:
Q7K, L33I, and L63I, which minimize the autoproteolysis
of the protease; and C67A and C95A, which prevent
cysteine-thiol oxidation. The kinetic parameters and stabil-
ity of this mutant are indistinguishable from those of the
mature enzyme [12,21]. Plasmid DNA (pET11a; Novagen)
encoding the PR was used, together with the appropriate
oligonucleotide primers, to generate the constructs PR
V82A
and PR
L90M
. All constructs were generated using the Quick-
Change mutagenesis protocol (Stratagene) and verified by
DNA sequencing and mass spectrometry. Escherichia coli
BL21(DE3) was grown in LB (Luria–Bertani) medium at
37 °C and induced for expression. Proteins were prepared
using an established protocol, as described previously [13].
Kinetic parameters, inhibition constants and urea
denaturation assay
The chromogenic substrate Lys-Ala-Arg-Val-Nle-p-nitro-
Phe-Glu-Ala-Nle-amide (Sigma) was used to determine the
kinetic parameters. Protease, at a final concentration of

70–120 n
M
, was added to varying concentrations of sub-
strate (25–400 l
M
), maintained in 50 m
M
sodium acetate,
pH 5.0, containing 0.1
M
NaCl, 1 m
M
EDTA, 1 m
M
2-mercaptoethanol, and assayed by monitoring the decrease
in absorbance at 310 nm using a PerkinElmer Lambda 35 or
a Hitachi U-3000 UV/Vis spectrophotometer. The absor-
bances were converted to substrate concentration via a
calibration curve. The enzyme concentrations were based on
active site titration data. The Michaelis–Menten curves were
fitted using
SIGMAPLOT
8.0.2 (SPSS Inc., Chicago, IL, USA).
The K
i
values were obtained from the 50% inhibitory
concentration (IC
50
) values, estimated from an inhibitor
dose)response curve with the spectroscopic assay and using

the following equation:
K
i
¼½ðIC
50
À½E=2Þ=ð1 þ½S=K
m
Þ
where [E] and [S] are the protease and substrate concentra-
tions, respectively [22].
The urea denaturation assay was carried out by measur-
ing the protease activity in the presence of 0–4.0
M
urea,
using the spectroscopic assay. Initial velocities were plotted
against urea concentration and fitted to a curve for solvent
denaturation using
SIGMAPLOT
8.0.2.
Crystallographic analysis
Crystals were grown at room temperature by vapor
diffusion using the hanging drop method. The protein
(5–10 mgÆmL
)1
) was preincubated with a 5- or 10-fold
molar excess of inhibitor. The crystallization drops con-
tained a 1 : 1 ratio, by volume, of reservoir solution and
protein. The final drop was typically 2 lL, and crystals grew
in 2–7 days. The wild-type and PR
V82A

crystals were
obtained using 0.05
M
citrate/phosphate buffer, pH 5.0–
5.6, with 0.2–0.4
M
NaCl as precipitant, while the PR
L90M
crystals were obtained using 0.1
M
sodium acetate buffer,
pH 4.6, with 2
M
ammonium sulfate as precipitant. The
crystals were frozen with a cryoprotectant of 20–30%
glycerol. X-ray diffraction data were collected on beamline
X26C at the National Synchrotron Light Source at
Brookhaven. Data were processed using the
HKL
suite
[23]. K45I (Protein Data Bank accession code 1DAZ)
protease coordinates were used as the starting model for
molecular replacement using
AMORE
[24]. The structures
were refined using
SHELX
[25] and modeled using O [26].
Alternate conformations for residues were modeled where
appropriate. The solvent was modeled with more than 150

water molecules, and ions were present in the crystallization
solutions. Anisotropic B factors were refined for all the
structures. Hydrogen atom positions were included in the
last stage of refinement using all data once all other
parameters, including disorder, had been modeled. The
structures have been submitted to the Protein Data Bank
with accession codes 1SDT(PR), 1SDU (PR
L90M
)and
1SDV (PR
V82A
). Crystal structures were superimposed on
all Ca atoms using an implementation of the algorithm
described previously [27].
Results and discussion
Kinetics and stability
The PR and two variants, PR
V82A
and PR
L90M
, harboring
single mutations that commonly appear in drug resistance,
including indinavir treatment [2,4], were chosen for this
study. While V82A alters a residue in the active site of
the protease that is critical to inhibitor binding, L90M is
distal to the inhibitor-binding site and is located near the
dimerization interface. The kinetic parameters of protease-
catalyzed hydrolysis of the chromogenic substrate, the
inhibition constants for the hydrolytic reaction by the
inhibitor indinavir, and the sensitivity to urea denaturation,

are shown in Table 1. PR exhibited the highest k
cat
value
compared to PR
V82A
and PR
L90M
, respectively. The k
cat
/K
m
values of PR
V82A
and PR
L90M
were % 85% and 81%,
respectively, relative to PR. The PR
L90M
hydrolysis of three
other peptide substrates has shown k
cat
/K
m
values of
% 40%, relative to PR, albeit under different assay condi-
tions [12]. The K
i
values for the three enzymes showed a
greater variation than the k
cat

/K
m
values. The apparent
Ó FEBS 2004 HIV protease crystal structures with indinavir (Eur. J. Biochem. 271) 1517
binding affinity of indinavir to PR
V82A
was about three-fold
lower than that of PR. Klabe et al. [28], using different assay
conditions, reported a six-fold lower K
i
value for the
PR
V82A
–indinavir interaction relative to that observed with
PR. The six-fold stronger inhibition of PR
L90M
by indinavir
is consistent with its enhanced inhibition by peptide analog
inhibitors [12]. The DDG values calculated for indinavir
binding were 0.79 and )1.19 kcal/mol for PR
V82A
and
PR
L90M
, respectively. These DDG values correspond to the
loss in PR
V82A
,orgaininPR
L90M
, of about one hydrogen

bond or van der Waals contact relative to the PR interaction
with indinavir.
The stability of PR
V82A
, as assessed by urea denaturation,
wassimilartothatofPR,whereasPR
L90M
was significantly
more sensitive to urea. In general, PR
V82A
,whichaltersa
residue in the inhibitor-binding site, showed similar activity
and stability, and lower inhibition, relative to PR. However,
the nonactive site mutant, PR
L90M
, had a slightly lower
activity, % 50% less stability, and improved inhibition by
indinavir. Therefore, the single mutations showed inde-
pendent effects on catalytic activity, inhibition, and stability,
consistent with our previous findings [12].
Crystal structures
The crystal structures are described for PR, PR
V82A
and
PR
L90M
in complexes with indinavir at 1.25–1.4 A
˚
resolution. These are the highest resolution complexes with
indinavir reported to date [17–20]. Data collection and

refinement statistics are given in Table 2. The crystallo-
graphic asymmetric unit had a dimer of HIV protease, with
the residues in the two subunits numbered 1–99 and 1¢)99¢.
In each structure the inhibitor was observed to have a single
orientation, and 168–183 water molecules were included.
The indinavir atoms are in well defined electron density,
with the exception of the pyridyl group that showed higher
B-values and disordered density, as shown for the PR
complex in Fig. 1. The solvent for the two structures in
space group P2
1
2
1
2 included two chloride ions, while the
L90M structure in space group P2
1
2
1
2
1
included a sulfate
and an acetate ion. All the ions were observed on the surface
of the protein.
Among the three complexes, the quality of the electron
density maps decreased in the order: PR>PR
L90M
>
PR
V82A
. The distribution of the mean B factors for the

main chain atoms showed similar peaks at the termini and
the variable surface loops of residues 16–18, 37–41, and 67–
69 of both subunits in all structures, except for the larger
values at 50–52 and 51¢)54¢ in PR, and 80–81 in PR
L90M
(Fig. 2). In PR
L90M
, the main chain atoms of residues 79–81
in one subunit have relatively high B factors and anisotropic
density. This anisotropy could be caused by the crystal
lattice because close van der Waals contacts (% 3.5 A
˚
)are
observed between Pro81 and symmetry related Tyr6¢ in
PR
L90M
. Although this region has similar crystal contacts in
both subunits, Pro79–Pro81 and the symmetry related Tyr6¢
are disordered, unlike the equivalent residues in the other
subunit. However, the benzyl ring of indinavir, which is
close to these residues, had very good electron density.
Higher main chain B factors were observed for flap residues
50–52 and 51¢)54¢ in PR relative to the values in the two
mutants. Ile50¢ was ordered in all three structures and the
side chain fitted well in the S2 binding pocket created by the
t-butyl group of indinavir. Ile50, on the other hand, was
disordered in all the structures and had poorer comple-
mentarity for the van der Waals interaction with the indanyl
group. Analysis of the anisotropic displacement parameters,
using

PARVATI
[29], showed a distribution typical for
Table 1. Kinetic data. Kinetic parameters for hydrolysis of the spectroscopic substrate (Lys-Ala-Arg-Val-Nle-p-nitroPhe-Glu-Ala-Nle-amide),
inhibition by indinavir and sensitivity to urea. The DDG values are calculated from RTlnKi. UC
50
, urea concentration at 50% activity.
Protease
K
m
(l
M
)
k
cat
(min
)1
)
k
cat
/K
m
(min
)1
Æl
M
)1
)
K
i
(p

M
)
DDG
(kcalÆmol
)1
)
UC
50
(
M
)
PR 55.0 ± 7.0 285.0 ± 9.5 5.2 ± 0.2 540 ± 70 0.00 1.95
a
PR
V82A
44.0 ± 6.5 194.5 ± 7.6 4.4 ± 0.1 1810 ± 270 0.79 1.77
PR
L90M
21.7 ± 1.9 91.2 ± 1.4 4.2 ± 0.4 86 ± 8 ) 1.19 1.00
a
a
Taken from Mahalingam et al. [12].
Table 2. Crystallographic data statistics. RMS, root mean square.
Protease mutant
PR PR
V82A
PR
L90M
Space group P2
1

2
1
2P2
1
2
1
2P2
1
2
1
2
1
Unit cell dimensions (A
˚
)
a 85.8 85.8 51.3
b 58.8 58.7 58.4
c 46.6 46.6 61.6
Unique reflections 54 269 41 646 50 225
R
merge
(%) 4.3 5.8 6.3
Overall (final shell) (25.8) (19.6) (37.4)
I/sigma(I) 28.68 24.35 23.14
Overall (final shell) (7.96) (8.17) (4.41)
Resolution range for
refinement (A
˚
)
10–1.30 10–1.40 10–1.25

R
work
(%) 15.51 15.97 14.11
R
free
(%) 19.04 20.54 18.32
No. of waters 169 183 168
Completeness (%) 92.7 88.7 96.8
Overall (final shell) (87.8) (91.5) (78.0)
RMS deviation from ideality
Bonds (A
˚
) 0.012 0.010 0.012
Angle distance (A
˚
) 0.029 0.028 0.030
Average B-factors (A
˚
2
)
Main chain 12.5 10.4 10.9
Side chain 17.5 14.5 16.5
Inhibitor 15.9 13.5 13.9
Solvent 26.1 24.7 25.8
1518 B. Mahalingam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
proteins with a mean anisotropy of 0.43–0.45 for protein
atoms. Atoms that display large anisotropy are typically
disordered or exhibit alternate conformations. The Cc and
Od2 atoms of Asp25 in PR and PR
V82A

, and both the Od
atoms of Asp25¢ in PR
L90M
, exhibited large anisotropy. This
anisotropy probably represents different states of charge
distribution and/or protonation of the catalytic aspartates.
Several residues showed disordered density for the side
chains and/or had alternate conformations (Table 3). Ile50,
Met46 and Met46¢ in the flaps were disordered in all three
structures. Disordered density has been reported previously
for hydrophobic protease residues that interact with
substrate analog inhibitors [13]. The side-chain of Val82
exhibited alternate conformations in both PR
L90M
and PR,
suggesting that its mobility is intrinsic. In PR, the alternate
conformations of Val82¢ appeared to be associated with
those of Arg8¢,Glu21¢ and two intervening water molecules.
This is an example of how residues that are further
away from the binding pocket can be associated with the
conformation of active-site residues. However, mutations of
Arg8 and Glu21 are rarely observed in resistant isolates [2].
One of the conformations of the side chain of Lys45 in PR
forms hydrogen bonds with the Od2 of Asp30, which
is expected to stabilize the flap. The other conformation of
Lys45 forms hydrogen bonds to the carbonyl oxygen of
Met46 through a water molecule.
ThesidechainsofMet90andMet90¢ in the PR
L90M
structure showed two conformations (Fig. 3), as described

previously for complexes with peptide analog inhibitors
[13,15]. Met90 showed one conformation with occupancy of
0.34, with the Ce atom at a short distance of 3.53 A
˚
from
the carbonyl oxygen of Asp25. The other conformation of
Met90, with 0.66 occupancy, had the Ce atom at 5.46 A
˚
from the carbonyl oxygen of Asp25. In the other subunit,
Met90¢ had one conformation at occupancy of 0.45 in which
the Ce atom was closer (3.43 A
˚
) to the carbonyl oxygen of
Asp25¢, while the other conformation had the Ce atom at
5.51 A
˚
from the carbonyl oxygen of Asp25¢. In comparison,
the Leu90 and Leu90¢ in the PR showed the closest distances
of 3.76 and 3.78 A
˚
to the carbonyl oxygen of the catalytic
Asp25 and Asp25¢, respectively. Alternate conformations
were not observed for Leu90 or Leu90¢ in either PR or
PR
V82A
. Therefore, the shorter van der Waals contact
between the minor conformation of Met90/90¢ and the
carbonyl oxygen of the catalytic Asp was proposed to
account for the lowered catalytic activity and stability of the
PR

L90M
mutant compared to the PR [13]. Similar close
contacts were reported for Met90 in the crystal structure of
the mutant G48V/L90M with saquinavir [30]. Presumably
the presence of the Met90 conformation in close contact
with Asp25 arises from an unusual electron distribution
around the catalytic residues that is required for the
proteolytic reaction.
Protease–indinavir interactions
One molecule of indinavir bound to protease residues from
both subunits (Table 4). The inhibitors in all the three
structures superpose very well, except for a small change in
position of the pyridyl end in PR
L90M
(Fig. 4A). The pyridyl
group of indinavir is accessible to the surface, while all the
other groups in indinavir are shielded either by a network of
water molecules or protease residues. Residues Arg8¢ and
Val82¢, which surround the partly disordered pyridyl group,
exhibit alternate side chain conformations in PR. The
Pro81¢ ring is puckered away to avoid unfavorable
interactions with C36 of the pyridyl group. Pro81, which
contacts the benzyl group, has a ring pucker towards the
Fig. 1. Omit map for indinavir in the wild-type HIV-1 protease (PR)
crystal structure. The contour level is 3.5. The polar atoms and pyridyl
group of indinavir are labeled.
Fig. 2. Mean B-values for main chain atoms of the wild-type HIV-1
protease (PR) and PR
L90M
. The mean B-values (A

˚
2
) are plotted for the
residuesofPR( )andPR
L90M
(––). The residues in the two subunits
are numbered 1–99 and 1¢)99¢.TheB-values for PR
V82A
are not shown
because they are lower than in the other structures.
Table 3. Amino acid side-chains with conformational flexibility.
Subunit
PR PR
V82A
PR
L90M
AB AB AB
Alternate
conformation
Glu21 Arg8¢, Met46¢ Met46
Lys45 Glu21¢ Val82¢
Ile33¢ Ile84
Val82 Val82 Met90 Met90¢
Disordered
density
Leu23 Lys45¢
Met46 Met46¢ Met46 Met46¢, Met46
Ile50 Phe53¢ Ile50 Ile50 Val82¢
Ó FEBS 2004 HIV protease crystal structures with indinavir (Eur. J. Biochem. 271) 1519
inhibitor. Thus, subtle conformational changes of residues

interacting with the inhibitor play a role in the kinetics.
The number of protease–indinavir van der Waals
contacts showed only a small variation among the three
crystal structures. PR had 96 van der Waals contacts,
with interatomic distances of < 4.0 A
˚
for the major
conformation of the side chains and 98 contacts for the
minor conformations. PR
V82A
showed 95 van der Waals
contacts with indinavir, similar to the PR. However,
PR
L90M
showed fewer van der Waals contacts with
indinavir than PR: 93 for the major and 92 for the minor
side chain conformations.
The three crystal structures showed a very similar
arrangement of proteaseÆindinavir hydrogen bond inter-
Fig. 3. Interaction of Met90¢ and Asp25¢ in PR
L90M
. (A) The 2Fo–Fc
electron density map showing Met90¢,Asp25¢ and Thr26¢ in the
PR
L90M
structure. The side chain of Met90¢ has two conformations,
and one conformation has a short separation from the carbonyl oxy-
gen of the catalytic Asp25¢.(B)ComparisonofMet90¢ in PR
L90M
and

Leu90¢ in the wild-type HIV-1 protease (PR) relative to Asp25¢.The
PR residues are in black and the PR
L90M
residues are in gray.
Hydrogen bonds are indicated by dashed lines, with the distances
shown in A
˚
.
Table 4. Protease residues with van der Waals interactions with indi-
navir. Interatomic distances of 3.3–4.2 A
˚
indicate van der Waals
contacts.
Subunit A Subunit B
Arg8
a
Arg8¢
b
Leu23 Leu23¢
c
Asp25
a
Asp25¢
a
Gly27
a
Gly27¢
a
Ala28 Ala28¢
Asp29¢

a
Asp30 Asp30¢
Val32 Val32¢
Ile47 Ile47¢
Gly48 Gly48¢
Gly49 Gly49¢
Ile50
a
Ile50¢
a
Pro81 Pro81¢
Val/Ala82 Val/Ala82¢
Ile84 Ile84¢
a
Residues with hydrogen bond or water–mediated interactions
(Fig. 4 and Table 5).
b
Hydrogen bond interaction only in PR
L90M
.
c
Interatomic distance of > 4.3 A
˚
in PR
L90M
.
Fig. 4. Protease hydrogen bond interactions with indinavir. The stereo
figures were prepared using
MOLSCRIPT
[31]. Hydrogen bonds are

indicated by dashed lines, with the distances shown in A
˚
.(A)Com-
parison of indinavir interactions with Arg8¢ in the wild-type HIV-1
protease (PR) (black) and PR
L90M
(gray). (B) PR interactions with
indinavir. The indinavir bonds are in black, the protease bonds are in
gray, and water molecules are represented as spheres and labeled A–D.
1520 B. Mahalingam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
actions, including the same water-mediated interactions,
except for one new interaction in PR
L90M
(Fig. 4 and
Table 5). Similar hydrogen bond and water-mediated
interactions were observed in the previous crystal structure
of wild-type protease with indinavir (PDB code 1HSG),
except that water OD was not observed [17]. Four water
molecules that mediate interactions between indinavir and
the protease were observed in all the high resolution crystal
structures. The O4 atom of indinavir formed hydrogen
bond interactions with the amide and OD2 of Asp29¢,and
interacted via water OB with the carbonyl oxygen of
Gly27, the OD1 of Asp29¢ and the NE atom of Arg8. The
N4 atom of indinavir interacted with the carbonyl oxygen
of Gly27¢. The indinavir O1 and O3 atoms formed
hydrogen bond interactions through a water molecule
(OA) to the amides of Ile50 and 50¢. Indinavir N2 showed
a water-mediated interaction with the amide nitrogen of
Asp29. Indinavir N1 formed a water-mediated hydrogen

bond to the carbonyl oxygen of Gly27; this interaction was
not observed in the 1HSG structure [17]. The O2 hydroxyl
group of indinavir formed hydrogen bonds with the four
carboxylate oxygens of Asp25 and 25¢. The four O2 to
carboxylate oxygen distances ranged from 2.7 to 2.9 A
˚
in
PR and PR
V82A
. However, PR
L90M
showed greater
asymmetry, with two shorter distances of 2.5 and 2.7 A
˚
and two longer distances of 2.9 and 3.2 A
˚
. It is possible
that the asymmetrical interaction of indinavir O2 with the
catalytic aspartates is associated with the short van der
Waals interaction of the Met90/90¢ side chains with the
carbonyl oxygen of Asp25/25¢ (Fig. 3B). In PR
L90M
,the
pyridyl N5 of indinavir formed a hydrogen bond with
the NH
2
of Arg8¢ (3.1 A
˚
). The corresponding distances in
PR and PR

V82A
were 4.0 and 4.2 A
˚
, respectively. This new
interaction could explain the better inhibition of PR
L90M
by indinavir.
Structural differences between mutants and PR
The PR
V82A
structure is very similar to the PR structure,
with a root mean square (RMS) deviation of 0.12 A
˚
for all
main chain atoms, as both crystal structures were obtained
in the same space group. Only the main chain atoms of
residues 81–82 and 81¢)82¢ showed larger RMS differences
of 0.34–0.59 A
˚
, while the catalytic triplets of 25–27 and
25¢)27¢ showed very low RMS differences, of 0.03–0.05 A
˚
for main chain atoms in both subunits (Fig. 5). The
estimated main chain errors (calculated from the B-values)
were 0.13–0.21 A
˚
for residues 81–82 and 81¢)82¢,and
0.08–0.12 A
˚
for the catalytic triplets. There was a small

movement of the main chain atoms of residues 81–82
towards indinavir in PR
V82A
, which partly compensated
for the change from Val82 in PR to the smaller side chain
of Ala (Fig. 6A). In PR, the two Cc atoms of Val82
formed van der Waals contacts with indinavir, of 3.8 A
˚
.In
PR
V82A
, the change in the position of the main chain
atoms placed the Cb atom of Ala82 within reasonable van
der Waals distance of indinavir (4.1 A
˚
), resulting in a loss
of only one contact compared to PR. In contrast, studies
of an inactive protease containing the mutations D25N/
V82A showed that Ala82 had no van der Waals contacts
with the drugs saquinavir or ritonavir [16]. The structural
changes observed in residues 81–82, which tend to
compensate for the smaller Ala82 compared to Val in
PR, were consistent with the small reduction in k
cat
/K
m
and the three-fold increased K
i
for indinavir with PR
V82A

relative to PR.
PR
L90M
showed an RMS difference of 0.61 A
˚
compared
to the PR, mainly owing to differences in lattice contacts in
the two space groups. The catalytic triplet residues 25–27
showed values of 0.08–0.15 A
˚
for comparison of main chain
atoms, consistent with the highly conserved core structure.
Differences of > 1.0 A
˚
were observed for the main chain
atoms of residues 16¢)18¢, 37–41/37¢)41¢,43¢)46¢,70¢)71¢
and 81 (Fig. 5). The estimated main chain errors were
0.37 A
˚
for Pro81 and 0.16–0.32 A
˚
for the other residues,
suggesting that differences of > 1.0 A
˚
are significant.
However, these large changes reflect variation in surface
residues owing to the different space groups, except for
Pro81 that forms part of the inhibitor-binding site. The
main chain atoms of residues 79–81 in PR
L90M

have
Table 5. Protease–indinavir hydrogen bond interactions.
Indinavir
Atoms Distance (A
˚
)
Water Protease PR PR
V82A
PR
L90M
1HSG
Direct interactions
O4 OD2 Asp29¢ 3.3 3.3 3.1 3.1
O4 N Asp29¢ 3.0 3.0 3.0 3.2
N4 O Gly27¢ 3.1 3.1 3.2 3.0
OH OD1 Asp25 2.8 2.8 2.9 2.8
OD2 Asp25 2.7 2.7 2.5 2.9
OD1 Asp25¢ 2.9 2.8 2.7 2.6
OD2 Asp25¢ 2.8 2.8 3.2 3.0
N5 NH2 Arg8¢ 3.1
Water-mediated interactions
O3 OA 2.6 2.7 2.6 2.8
O1 OA 2.8 2.8 2.8 2.7
OA N Ile50 3.0 2.9 2.9 3.1
OA N Ile50¢ 2.9 2.9 2.9 2.9
O4 OB 3.3 3.2 3.3 2.8
OB O Gly27¢ 2.7 2.7 2.6 3.2
OB OD1 Asp29¢ 2.8 2.8 2.8 2.7
OB NE Arg8 3.1 3.1 3.1 2.8
N2 OC 3.1 3.1 3.0 3.3

OC N Asp29 2.9 2.9 2.9 3.0
N1 OD 3.0 3.1 3.0
OD O Gly27 3.1 2.9 2.9
Fig. 5. Structural differences in main chain atoms. The root mean
square (RMS) differences (A
˚
) per residues are plotted for main chain
atoms of PR
V82A
(––) and PR
L90M
(- - -) compared with the wild-type
HIV-1 protease (PR).
Ó FEBS 2004 HIV protease crystal structures with indinavir (Eur. J. Biochem. 271) 1521
anisotropic electron density. The density is ordered in the
plane of the interaction with indinavir, shown in Fig. 6B,
and extended/disordered in the perpendicular direction.
Although the main chain atoms of 80–81 in PR
L90M
have
moved 0.6–1.2 A
˚
further from indinavir compared to PR,
the Cc of P81 has maintained similar van der Waals
contacts with C19 of indinavir (3.8 and 3.9 A
˚
in PR and
PR
L90M
, respectively) (Fig. 6B). The closest Cc atom of

Val82 is 4.1 A
˚
from indinavir, only a little farther than the
3.9 A
˚
separation in PR. The positions of the main chain
atoms, of 80–82, relative to indinavir, were consistent with
the smaller number of van der Waals contacts between the
protease and indinavir observed for PR
L90M
compared to
PR (93 compared to 96).
Residues 81¢ and 82¢ interact with the pyridyl group of
indinavir. There appear to be small correlated changes in the
position of the side chain of Pro81¢, the pyridyl group, Arg8¢
and Phe53 in PR
L90M
, relative to their positions in PR and
PR
V82A
. The carboxylate groups of the catalytic Asp25 and
Asp25¢ also showed a small shift relative to their positions in
the other complexes, and less symmetrical interactions with
the hydroxyl of indinavir (Table 5), as well as the close
contacts between the carbonyl oxygen atoms and Met90/90¢
(Fig. 3). It is probable that these small structural changes
result in the lowered activity and stability of PR
L90M
relative
to PR (Table 1). The increase in affinity for indinavir may

arise from the new hydrogen bond interaction between the
pyridyl of indinavir and Arg8¢, and the structural changes
associated with the close contact between Met90/190 and
the catalytic aspartates. The new hydrogen bond interaction
is consistent with the observed DDGof)1.19 kcal/mol for
the inhibition of PR
L90M
compared to PR.
The structural changes described for PR
V82A
and
PR
L90M
, relative to PR, differ from those reported in
previous studies with other mutations. Munshi et al.[19]
suggested that the 80 s loop is intrinsically flexible; however,
mutations can influence the conformation of this loop and
its interactions with indinavir. PR with mutations M46I/
L63P/V82T/I84V showed structural changes in the flaps
near the mutated Ile46, and in the interactions of the
mutated Thr82 and Val84 with indinavir [18]. Local changes
in the mutated residues were also observed in the crystal
structure of the L63P/V82T/I84V mutant with indinavir
[20]. Our structures of PR
V82A
and PR
L90M
showed
opposite changes in the main chain atoms of residues
81–82, and PR

L90M
also had changes in the conformation of
the catalytic aspartates, probably associated with the close
contact of Met90, and in the side chains interacting with the
pyridyl group of indinavir. The mutation V82A produced
local changes around residue 82, while L90M showed both
local and more distal changes propagating to the inhibitor-
binding site.
Conclusions
The optimized wild-type HIV-1 protease (PR) and the drug-
resistant mutants, PR
V82A
and PR
L90M
, were compared by
using crystallographic and kinetic analysis. The two mutants
showed slightly decreased k
cat
/K
m
values as compared to
PR. PR
V82A
and PR
L90M
had increased and decreased K
i
values for indinavir, respectively, compared to PR. Most of
the interactions with indinavir were similar for the three
high resolution crystal structures. Small differences were

observed in the van der Waals contacts with indinavir for
the mutants compared to PR. The active site mutant,
PR
V82A
, showed changes in the positions of the main chain
atoms of residues 81–82 in both subunits that partially
compensated for the mutation by improved interactions
with indinavir. In contrast, PR
L90M
showed fewer van der
Waals contacts with indinavir, the main chain atoms of
residues 80–82 were further from the indinavir, and the side
chain of Met90 and Met90¢ had altered interactions with the
catalytic Asp25 and Asp25¢. However, there is a new polar
interaction between the pyridyl N5 of indinavir and the side
chain of Arg108, which may account for the apparent
decreased K
i
of PR
L90M
for indinavir. Both the mutants
showed small structural changes around the indinavir that
must be interpreted, together with kinetic and stability data,
in order to understand the effect of the mutation. The lower
stability of PR
L90M
is consistent with the observed small
structural changes in Asp25 and Asp25¢ at the dimer
interface. The DDG values for binding of indinavir corres-
pond to the observed loss in PR

V82A
of about one van der
Waals contact and gain in PR
L90M
of one hydrogen bond
relative to the PR interaction with indinavir. The structural
and kinetic data suggest that the resistant mutation, V82A,
acts directly to reduce the affinity for indinavir, while L90M
appears to act indirectly by lowering the dimer stability,
despite the apparent higher affinity for indinavir. The
changes in protease structure and interactions with indinavir
must be considered during the design of new inhibitors for
resistant HIV.
Fig. 6. Structural variation in residues 81–82 near indinavir. Stereoview
showing the benzyl group of indinavir interacting with residues 81–82,
using the major conformation of Val82. The wild-type HIV-1 protease
(PR) structure is in black and the mutant is in gray bonds. Interatomic
distances are given in A
˚
.(A)PR
V82A
superimposed on PR. (B) PR
L90M
superimposed on PR.
1522 B. Mahalingam et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Acknowledgements
We thank Xianfeng Chen for assistance with discussion of protease–
indinavir interactions. We thank Merck & Co. for providing the
indinavir used for the crystallographic analysis. The X-ray diffraction
data were recorded at the beamline X26C of the National Synchrotron

Light Source at Brookhaven National Laboratory, which is supported
by the US Department of Energy, Division of Materials Sciences and
Division of Chemical Sciences, under Contract No. DE-AC02-
98CH10886. The research was supported, in part, by the Georgia
Research Alliance, National Institute of Health grants GM62920,
AIDS-FIRCA TW01001, and Hungarian OTKA F35191 and T43482.
References
1. Hertogs, K., Bloor, S., Kemp, S.D., Van den Eynde, C.,
Alcorn, T.M., Pauwels, R., Van Houtte, M., Staszewski, S.,
Miller,V.&Larder,B.A.(2000)Phenotypicandgenotypic
analysis of clinical HIV-1 isolates reveals extensive protease in-
hibitor cross-resistance: a survey of over 6000 samples. AIDS 14,
1203–1210.
2. Wu, T.D., Schiffer, C.A., Gonzales, M.J., Taylor, J., Kantor, R.,
Chou, S., Israelski, D., Zolopa, A.R., Fessel, W.J. & Shafer, R.W.
(2003) Mutation patterns and structural correlates in human
immunodeficiency virus type 1 protease following different pro-
tease inhibitor treatments. J. Virol. 77, 4836–4847.
3. Condra, J.H., Holder, D.J., Schleif, W.A., Blahy, O.M., Danov-
ich, R.M., Gabryelski, L.J., Graham, D.J., Laird, D., Quintero,
J.C., Rhodes, A., Robbins, H.L., Roth, E., Shivaprakash, M.,
Yang,T.,Chodakewitz,J.A.,Deutsch,P.J.,Leavitt,R.Y.,Mas-
sari, F.E., Mellors, J.W., Squires, K.E., Steigbigel, R.T., Teppler,
H. & Emini, E.A. (1996) Genetic correlates of in vivo viral
resistance to indinavir, a human immunodeficiency virus type 1
protease inhibitor. J. Virol. 70, 8270–8276.
4. Sa-Filho, D.J., Costa, L.J., de Oliveira, C.F., Guimaraes, A.P.,
Accetturi, C.A., Tanuri, A. & Diaz, R.S. (2003) Analysis of the
protease sequences of HIV-1 infected individuals after indinavir
monotherapy. J. Clin. Virol. 28, 186–202.

5. Brown, A.J., Korber, B.T. & Condra, J.H. (1999) Associations
between amino acids in the evolution of HIV type 1 protease
sequences under indinavir therapy. AIDS Res. Hum. Retroviruses
15, 247–253.
6. Zennou,V.,Mammano,F.,Paulous,S.,Mathez,D.&Clavel,F.
(1998) Loss of viral fitness associated with multiple Gag and Gag-
Pol processing defects in human immunodeficiency virus type 1
variants selected for resistance to protease inhibitors in vivo.
J. Virol. 72, 3300–3306.
7. Louis, J.M., Weber, I.T., Tozser, J., Clore, G.M. & Gronenborn,
A.M. (2000) HIV-1 protease: maturation, enzyme specificity, and
drug resistance. Adv. Pharmacol. 49, 111–146.
8. Gulnik, S.V., Suvorov, L.I., Liu, B., Yu B., Anderson, B., Mits-
uya, H. & Erickson, J.W. (1995) Kinetic characterization and
cross-resistance patterns of HIV-1 protease mutants selected under
drug pressure. Biochemistry 34, 9282–9287.
9. Ermolieff, J., Lin, X. & Tang, J. (1997) Kinetic properties of
saquinavir-resistant mutants of human immunodeficiency virus
type 1 protease and their implications in drug resistance in vivo.
Biochemistry 36, 12364–12370.
10. Ridky, T.W., Kikonyogo, A., Leis, J., Gulnik, S., Copeland, T.,
Erickson, J., Wlodawer, A., Kurinov, I., Harrison, R.W. &
Weber, I.T. (1998) Drug-resistant HIV-1 proteases identify
enzyme residues important for substrate selection and catalytic
rate. Biochemistry 37, 13835–13845.
11. Xie, D., Gulnik, S., Gustchina, E., Yu B., Shao, W., Qoronfleh,
W., Nathan, A. & Erickson, J.W. (1999) Drug resistance
mutations can effect dimer stability of HIV-1 protease at neutral
pH. Protein Sci. 8, 1702–1707.
12. Mahalingam,B.,Louis,J.M.,Reed,C.C.,Adomat,J.M.,Krouse,

J., Wang, Y.F., Harrison, R.W. & Weber, I.T. (1999) Structural
and kinetic analysis of drug resistant mutants of HIV-1 protease.
Eur. J. Biochem. 263, 238–245.
13. Mahalingam, B., Louis, J.M., Hung, J., Harrison, R.W. &
Weber, I.T. (2001) Structural implications of drug resistant
mutants of HIV-1 protease: high resolution crystal structures
of the mutant protease/substrate analog complexes. Proteins 43,
455–464.
14. Feher, A., Weber, I.T., Bagossi, P., Boross, P., Mahalingam, B.,
Louis, J.M., Copeland, T.D., Torshin, I.Y., Harrison, R.W. &
Tozser, J. (2002) Effect of sequence polymorphism and drug
resistance on two HIV-1 Gag processing sites. Eur. J. Biochem.
269, 4114–4120.
15. Mahalingam, B., Boross, P., Wang, Y F., Louis, J.M., Fischer,
C., Tozser, J., Harrison, R.W. & Weber, I.T. (2002) Combining
mutations in HIV-1 protease to understand mechanisms of
resistance. Proteins 48, 107–116.
16. Prabu-Jeyabalan, M., Nalivaika, E.A., King, N.M. & Schiffer,
C.A. (2003) Viability of a drug-resistant human immunodeficiency
virus type 1 protease variant: structural insights for better antiviral
therapy. J. Virol. 77, 1306–1315.
17. Chen, Z., Li, Y., Chen, E., Hall, D.L., Darke, P.L., Culberson, C.,
Shafer, J.A. & Kuo, L.C. (1994) Crystal structure at 1.9 angstroms
resolution of human immunodeficiency virus (HIV) II protease
complexed with l-735,524, an orally bioavailable inhibitor of the
HIV proteases. J. Biol. Chem. 269, 26344–26348.
18. Chen, Z., Li, Y., Schock, H.B., Hall, D., Chen, E. & Kuo, L.C.
(1995) Three-dimensional structure of a mutant HIV-1 protease
displaying cross-resistance to all protease inhibitors in clinical
trials. J. Biol. Chem. 270, 433–436.

19. Munshi, S., Chen, Z., Yan, Y., Li, Y., Olsen, D.B., Schock, H.B.,
Galvin, B.B., Dorsey, B. & Kuo, L.C. (2000) An alternate binding
site for the P1–P3 group of a class of potent HIV-1 protease
inhibitors as a result of concerted structural change in the 80s loop
of the protease. Acta Crystallogr. D56, 381–388.
20. King, N.M., Melnick, L., Prabu-Jeyabalan, M., Nalivaika, E.A.,
Yang,S.S.,Gao,Y.,Nie,X.,Zepp,C.,Heefner,D.L.&Schiffer,
C.A. (2002) Lack of synergy for inhibitors targeting a multi-drug-
resistant HIV-1 protease. Protein Sci. 11, 418–429.
21. Louis, J.M., Clore, G.M. & Gronenborn, A.M. (1999) Auto-
processing of HIV-1 protease is tightly coupled to protein folding.
Nat. Struct. Biol. 6, 868–875.
22. Maibaum, J. & Rich, D.H. (1988) Inhibition of porcine pepsin by
two substrate analogues containing statine: the effect of histidine
at the P2 subsite on the inhibition of aspartic proteinases. J. Med.
Chem. 31, 625–629.
23. Otwinowski, Z. & Minor, W. (1997) Processing of X-ray diffrac-
tion data in oscillation mode. Methods Enzymol. 276, 307–326.
24. Navaza, J. (1994) AMoRe: An automated package for molecular
replacement. Acta Crystallogr. D50, 157–163.
25. Sheldrick, G.M. & Schneider, T.R. (1997) High resolution
refinement. Methods Enzymol. 277, 319–343.
26. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. (1991)
Improved methods for building protein models in electron density
maps and the location of errors in these models. Acta Crystallogr.
A47, 110–119.
27. Ferro, D.R. & Hermans, J. (1977) A different best rigid-body
molecular fit routine. Acta Crystallogr. A33, 345–347.
28. Klabe, R.M., Bacheler, L.T., Ala, P.J., Erickson-Vitanen, S. &
Meek, J.L. (1998) Resistance to HIV protease inhibitors: a

comparison of enzyme inhibition and antiviral potency. Bio-
chemistry 37, 8735–8742.
Ó FEBS 2004 HIV protease crystal structures with indinavir (Eur. J. Biochem. 271) 1523
29. Merritt, E.A. (1999) Expanding the model: anisotropic displace-
ment parameters in protein structure refinement. Acta Crystallogr.
D55, 1109–1117.
30. Hong, L., Zhang, X.C., Hartsuck, J.A. & Tang, J. (2000) Crystal
structure of an in vivo HIV-1 protease mutant in complex
with saquinavir: insights into the mechanisms of drug resistance.
Protein Sci. 9, 1898–1904.
31. Kraulis, P.J. (1991)
MOLSCRIPT
: a program to produce both
detailed and schematic plots of protein structures. J. Appl. Cryst.
24, 946–950.
1524 B. Mahalingam et al. (Eur. J. Biochem. 271) Ó FEBS 2004