Tài liệu Báo cáo khoa học: Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors docx

Role of hydroxyl group and
R
/
S
configuration of isostere in binding
properties of HIV-1 protease inhibitors
Hana Petrokova
´
1
, Jarmila Dus
ˇ
kova
´
1
, Jan Dohna
´
lek
1
, Tereza Ska
´
lova
´
1
, Eva Vondra
´
c
ˇ
kova
´
-Buchtelova
´

1
,
Milan Souc
ˇ
ek
2
, Jan Konvalinka
2
, Jir
ˇ
ı
´
Brynda
3
, Milan Fa
´
bry
3
, Juraj Sedla
´
c
ˇ
ek
3
and Jindr
ˇ
ich Has
ˇ
ek
1

1
Institute of Macromolecular Chemistry,
2
Institute of Organic Chemistry and Biochemistry and
3
Institute of Molecular Genetics,
Academy of Sciences of the Czech Republic, Praha, Czech Republic
The crystal structure of the complex between human
immunodeficiency virus type 1 (HIV-1) protease and a
peptidomimetic inhibitor of ethyleneamine type has b een
refined to R factor of 0.178 w ith diffraction limit 2.5 A
˚
.The
peptidomimetic inhibitor Boc-Phe-Y[CH
2
CH
2
NH]-Phe-
Glu-Phe-NH
2
(denoted here as OE) c ontains the ethylene-
amine replacement of the s cissile peptide bond. The inhibitor
lacks the hydroxyl group which is believed to mimic tetra-
hedral transition state o f p roteolytic reaction and thus is
suspected to be necessary for g ood properties of p eptido-
mimetic HIV-1 protease inhibitors. Despite the missing
hydroxyl group the inhibition constant of OE is 1.53 n
M
and it remains in the nanomolar range also towards several
available mutan ts of HIV-1 protease. The inhibitor was

found in the active site of protease in an extended
conformation with a unique hyd rogen bond pattern different
from hydroxyethylene and hydroxyethylamine i nhibitors.
The isostere n itrogen forms a hydrogen bond to one catalytic
aspartate only. The other aspartate forms two weak h ydro-
gen bridges to the e thylene group of the isostere. A com-
parison with o ther inhibitors of this series containing isostere
hydroxyl group in R or S configuration shows different w ays
of accommodation of inhibitor in the active site. Special
attention is devoted to intermolecular contacts between
neighbouring dimers responsible for mutual protein adhe-
sion and for a special conformation of Met46 a nd Phe53 s ide
chains not expected for free p rotein in water solution.
Keywords: ethyleneamine inhibitor; HIV-1 protease; pepti-
domimetic inhibitor; X-ray s tructure.
The HIV-1 protease, the aspartic protease that cleaves
specific peptide bonds in precursor gag-pol proteins to form
the mature proteins, is essenti al for production of infectious
HIV p articles. Its inhibition is an efficient method of
treatment of the acquired immunodeficiency syndrome
(AIDS) and r elated diseases [1,2]. No matter what is the
inhibitor type (symmetrical or unsymmetrical), the H IV
protease covers any i nhibitor under its flaps (Fig. 1) forming
thus a characteristic long binding tunnel with the cleavage
site in the m iddle. One structural f eature present in m ost
tight-binding aspartic protease inhibitors is a critical
hydroxyl gro up that replaces the catalytical water molecule
in the active site. This hydroxyl group forms hydrogen
bonds to the catalytically active aspartates [3] in HIV
protease complexes with all hydroxyethylamine inhibitors

being by f ar the m ost frequently studied str uctures in the
Protein Data Bank [ 4] an d the HIV P rotease Database
( and therefore it has been
supposed necessary for tight-binding of aspartic prote ase
inhibitors.
This paper presents the structure of native HIV-1
protease in a complex with inhibitor Boc-Phe-
Y[CH
2
CH
2
NH]-Phe-Glu-Phe-NH
2
(denoted here as OE).
The inhibition constant of OE remains low (1.53 n
M
)in
spite of the fact that the critical hydroxyl group is
completely miss ing i n this i nhibitor a nd it remains in the
nanomolar range also for several available m utants of HIV-
1 protease (e.g. 4.1 n
M
for the A71V/V82T/I84V mutant
arising after Indinavir treatment) [5,6].
The s tructure of OE complex has been solved in the frame
of a systematic structure study of a group of inhibitors with
very similar chemistry. They differ only in the presence of
the hydroxyl group in the isostere and in its configuration
(R or S) which is considered crucial for tight binding of
hydroxyethylamine inhibitors. The fact that the inhibition

constant of the respective inhibitors (denoted here as OE,
RE and SE) does not differ much deserves closer attention.
The inhibitor OE (without OH group) has only a slightly
lower inhibition efficiency than similar i nhibitors possessing
hydroxyl group in S or R configuration ( K
i
,
OE
¼ 1.5 n
M
,
K
i
,
RE
¼ 0.12 n
M
, K
i
,
SE
¼ 0.15 n
M
) [5,7–9].
Our recent studies of the hydroxyethylamine inhibitor
complexes [ 7–9] revealed that the binding tunnel of p rotease,
abundant in hydrogen bond donors and acceptors, can
bind the inhibitors i n several possible ways. Most of the
Correspondence to H. Petrokova
´

, Institute of Macromolecular
Chemistry, Academy of Sciences of the Czech Republic, Heyrovske
´
ho
na
´
m. 2, 162 06 Praha 6. Fax: + 420 296809 410,
Tel.: +420 296809 205, E-mail:
Abbreviations: OE, Boc-Phe-Y[CH
2
CH
2
NH]-Phe-Glu-Phe-NH
2
;RE,
Boc-Phe-Y[(R)-CH(OH)CH
2
NH]-Phe-Glu-Phe-NH
2
;SE,
Boc-Phe-Y[(S)-CH(OH)CH
2
NH]-Phe-Glu-Phe-NH
2
.
Enzyme: retropepsin (EC 3.4.23.16).
Note: The crystallographic data o f the complex HIV-1 protease with
OE in hibitor have been deposited with the P rotein Data Bank and a re
available u nder access code 1m0b.
(Received 2 1 June 2004, re vised 9 September 2004,

accepted 29 September 2004)
Eur. J. Biochem. 271, 4451–4461 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04384.x
hydroxyethylene inhibitors are bound to the HIV)1
protease by interaction of the hydr oxyl gr oup o f i sostere
with both aspartates Asp25, Asp125. A slight shift (about
0.5 A
˚
) of t he isostere group in the c ase o f h ydroxyethyl-
amine inhibitors SE or SQ [7,8] c auses t hat the catalytic
aspartates bind mainly to the isostere NH group leaving
only one contact to the isostere hydroxyl group. The NH
group thus p artly substitutes t he role of the hydroxyl group
in hydroxyethylene inhibitors. In this paper, we present
another unusual binding mode of the ethyleneamine
inhibitor OE where only its isostere NH group makes a
contact to one catalytic aspartate. We suggest that the
binding mode where the role of the hydroxyl g roup is
completely overtaken by the isostere N H group can b e
generalized for the who le class of ethyleneamine inhibitors.
Materials and methods
Crystallization and crystal parameters
A solution of HIV protease at concentration 3 mgÆmL
)1
in 50 m
M
sodium acetate buffer (pH 5.8) containing 0.5%
(v/v) 2-mercaptoethanol was mixed with the inhibitor [12]
dissolved in dimethyl sulfoxide at 11 m
M
in the volume r atio

20 : 1 and left at 4 °C for at least 30 min prior to
crystallization; this gave the final fourfold molar excess of
inhibitor [11]. Co-crystallization by hanging drop diffusion
technique against 1 mL reservoir of 0.2–0.6
M
NaCl in
0.1
M
Na citrate buffer, pH 4.5–5.5 followed. Rod-shaped
hexagonal crystals appeared overnight a nd continued to
grow over the n ext 7 days. B efore flash-freezing, the crystals
were soaked for 30 s in the mother liquor containing 20%
glycerol.
Data collection and processing
X-ray diffraction data were collected at the European
Synchrotron R adiation Facility (ESRF) in Grenoble at
BM29 beamline equipped with a MAR 345 detector.
Data were collected from a single crystal (0.06 ·
0.06 · 0.7 mm) at a temperature of 100 K. The oscilla-
tion range was 1.5° and each frame was exposed for
30 s. The distance of the crystal to detector plate was
100 mm. The diffraction data ex tended t o 2 .0 A
˚
.Inten-
sities were integrated, scaled and merged using the
HKL
software [12]. D ata were r educed in the P 6
1
space group.
The unit cell dimensions were a ¼ b ¼ 62.7 A

˚
, c ¼
82.2 A
˚
. The details of X-ray diffraction data collection
are described in Table 1.
Refinement
Refinement was carried out using the
CNS
program
package [ 13]. Parameters for nonstandard parts of
inhibitor were set in agreement with several structures
found in the CCDC database [14]. The rigid body
refinement was performed with the starting model o f the
protease dimer taken from the Protein Data Bank [4]
(PDB code 1aaq). Several cycles of
CNS
refinement
(positional and individual B factor optimization) and
rebuilding using the graphics program
O
[15] were carried
out. The noncrystallographic symmetry was applied
during the refinement to both the protease and the
inhibitor at the initial stages of refinement with the
weight of 300 kcalÆmol
)1
ÆA
˚
)2

. Later, it was partially
Fig. 1. Fron t view of the structure of n ative HIV-1 protease complexed
with OE inhibitor. The inhibitor (stick model) sits over the catalytic
aspartates (ball- and-stick model) and is completely covered b y prote-
ase fl aps belonging t o two monomers of p rotease related by an
approximate two-fold s ymmetry axis. The exact C
2
symmetry is per-
turbed by asymmetry of the inhibitor and also by contacts between
neighbouring protease subunits.
Table 1. S tatistics of d iffracted intensity measurement. Complex o f
native HIV-1 protease with inhibitor OE. R
sym
¼ S|I ) <I>|/
S<I>.
All reflections The highest shell
Diffraction limits (A
˚
) 25–2.45 2.51–2.45
No. of observed reflections 42257 3548
No. of unique reflections 6765 865
R
sym
0.091 0.495
Completeness (%) 99.2 98.5
I/r
I
14.5 3.14
Mosaicity (deg.) 0.495 –
Table 2. P arameters describing the quality of refined m odel of t he native

HIV-1 protease c omplexed with inhibitor OE.
Parameter
No. of non-H protein atoms 1516
No. of non-H inhibitor atoms 50
No. of refined water oxygens 159
R factor (all reflections) 0.18
R factor (working set of reflections) 0.18
R free (5% of randomly selected reflexions) 0.24
rmsd from ideal bond lengths (A
˚
) 0.011
rmsd from ideal bond angles (°) 1.8
% of cases in the most favored
regions of Ramachandran plot
92.4
% of cases in disallowed regions
of Ramachandran plot
0
average B for main chain atoms (A
˚
2
) 30.1
average B for side chain atoms (A
˚
2
) 31.5
4452 H. Petrokova
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2004
liberated to 50 kcalÆmol

)1
ÆA
˚
)2
. Cross validation was
performed with the use of the R
free
factor calculated
from 5% of the reflections.
The i nhibitor was uniquely identified and modeled in the
2F
o
-F
c
map in its expected position in two opposite
orientations at R ¼ 0.256 and R
free
¼ 0.2 94. After the
structure refinement to R ¼ 0.235 and R
free
¼ 0.272, a total
of 159 water m olecules w ere gradually included. The criteria
for accepting waters in refinement were as follows: the
presence of the F
o
-F
c
electron density peak at 3 r level, the
2F
o

-F
c
peak at 1 r level, and at least one hydrogen-bonding
partner within the distance 2.2–3.6 A
˚
.
The resulting structure (Fig. 1) has R ¼ 0.178 and
R
free
¼ 0.242. Parameters d escribing the quality of the final
structure were checked b y program
PROCHECK
[16] and are
given in T able 2. The symmetry of the com plex and rigidity
of different parts of the protease can be seen in Figs 2,3. The
figures were produced using programs
MOLSCRIPT
[17] and
RASTER
3
D
[18] or
WLVIEWERPRO
( />weblab).
Results
Structure of the complex of native HIV-1 protease
with OE inhibitor
The s tructure of the HIV-1 proteas e in the complex with
inhibitor OE (Fig. 1) has b een finally refined to R ¼ 0.178
and R

free
¼ 0.242. The protein is a homodimeric molecule,
made up of two 99-residue polypeptide chains: chain A,
Pro1-Phe99 and chain B, Pro101-Phe199. The inhibitor is
bound as an extended c hain in a tunnel running under fl aps
across the dimer interface. The flaps consist of the amino-
acid residues 46–55 and 146–155 from both polypeptide
chains and completely close the inhibitor in the active site
channel. The tips of flaps bind together by hydrogen bond
(NH Gly51…C ¼ O Ile150 or NH Gly151…C ¼ O I le50).
The nitrogen atoms of Ile50 and Ile150 in flaps interact with
the inhibitor via hydrogen bonding through a single water
molecule (W401) typically present in s tructures o f H IV-1
protease with peptidomimetic inhibitors.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
11
21
31
41 51 61
71 81
91
1

Residue number
Fig. 2. Root-m ean-square distances between
the corresponding atoms of individual residues
after the le as t-squ ares alignment of C
a
atoms o f
two subunits A and B of the complex of HIV-1
protease with OE inhibitor.
0
20
40
60
80
0
20
40
60
80
21111 31415161718191
Residue number
chain A
chain B
B factor
B factor
Fig. 3. The complex of HIV-1 protease with
OE inhibitor B fa ctors averaged o ver each
residue. Top, c hain A; bottom, chain B.
Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur. J. Biochem. 271) 4453
Inhibitor binding. The inhibitor OE b inds into the p rotease
binding tunnel in an e xtended c onformation in two opposite

directions (Fig. 4). The flaps are locked over the inhibitor by
water molecule W401 forming hydrogen bonds to NH
groups of Ile50, Ile150 and to carbonyls of the inhibitor in
positions P
1
¢ and P
2
(Fig. 5). The side chains of t he tert-
butyloxycarbonyl (P
2
), phenylalanine (P
1
), phenylalanine
(P
1
¢), glutamine (P
2
¢) and phenylalaninamide (P
3
¢)bindin
the respective S
2
,S
1
,S
1
¢,S
2
¢ and S
3

¢ pockets of the HIV-1
protease (Fig. 6).
The inhibito r was modelled into a well defined F
o
-F
c
electron density after the protein was refined to R ¼ 0.25
and R
free
¼ 0.29. The disordered i nhibitor was modelled in
the a ctive site of protease in two opposite orientations
(Fig. 4), here referred as I a nd Y chains (residue numbers in
PDB file 301A-306A and 301B-306B, respectively). The
average rmsd of inhibitors in opposite orientations ( I a nd Y)
was 0.15 A
˚
.
The s cheme o f h ydrogen bonds (Fig. 5) shows t hat a ll
proton donors and acceptors of the inhibitor are involved in
hydrogen bonding. Five h ydrogen bonds of the total 18
Fig. 4. Stere oview of two alternative positions of the inhi bitor OE i n the el ectron density 2F
o
-F
c
at 1 r level. Both orientations were refined with
occupation factors 0.5. In b all-and-stick model the a ctive site Asp25–Gly27 and Asp125–Gly127 are highlighted.
N
O
N
O

N
O
O
O
N
O
O
O
N
OH
O
N
N
O
N
O
O
N
O
O
N
O
O
N
N
O
N
O
O
O

N
O
O
OH
3.5
Asp125
Gly127
Asp25
Gly27
Gly48
3.6
2.7
2.5
3.2
W401
3.3
W402
3.1
W464
2.4
3.5
Asp29
Asp30
W424
W507
3.3
3.1
2.8
3.0
3.3

2.6
3.0
3.3
2.9
Ile50
Ile150
Fig. 5. Network of hydrogen bonds formed by
the inhibitor OE in the native HIV-1 protease.
Distances are given in A
˚
. Statistics: one
intramolecular hydrogen bond CO…NH,
seven hydrogen bonds to protease main-chain
NH, three to side-ch ain carboxyls, three to
main-chain carbonylsandseventowater
molecules.
D129
G148
G149
F153
R8
N
O
O
N
O
N
O
OH
O

N
O
N
N
O
O
O
D30
A28
V32
I150
I47
G48
I84
G149
I150
P81
V82
I84
D25
T80
G127
A128
D130
V132
I147
D129
G148
G49
V182

L123
P181
G27
G48
I50
D25
S2
S1
S1'
S2'
S3'
Fig. 6. Rev iew of hydrophobic interactions (short C-C contacts up to
4.1 A
˚
) of t he inhibitor OE side chains with binding pockets of HIV-1
protease.
4454 H. Petrokova
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2004
hydrogen bonds connect directly the inhibitor main chain
with the main chain of protease (NH…CO to Gly23,
Gly123, Gly48, CO…NH to Gly48, Asp29). Two hydrogen
bonds connect the side chain of the inhibitor (Glu) with the
protein m ain c hain (CO …NH to Asp29, Asp30). T hree
hydrogen bonds connect inhibitor with protease s ide chains
(NH…Asp125, COOH…Asp30, NH
2
…Asp30). Three
structurally important water m olecules W 401, W402 and
W464 bridge directly the inhibitor with protease an d form

five hydrogen bonds to inhibitor P
2
(CO…W402), P
1
(NH…W402 and CO…W401), P
0
1
(CO…W401), P
0
2
(CO…W464). The whole inhibitor is almost completely
buried in the binding t unnel o f p rotease. Only the inhibitor
ends seem to be exposed to solvent. Two water molecules
W424 and W507 were found in the difference map form ing
two h ydrogen b ridges to the N terminal group of inhibitor.
Unusual conformation of isostere in the inhibitor OE
enables also a weak intramolecular hydrogen bond con-
necting the Boc carbonyl w ith the NH group at P
1
¢ position
(O…Nat3.6A
˚
). The inhibitor refined in the opposite
orientation binds in a similar way.
The inhibitor G lu at P
2
¢ is totally buried i n the protease
S
2
¢ s ite an d makes six h ydrogen bonds to Asp30 and Asp29.

The importance of this r esidue and the strength of its
binding to protease are supported also by t he fact that it has
the lowest B-factors of the whole inhibitor.
The w ater molecule W401 that hydrogen bonds to the
main chain NH group of both flaps as well as to the
inhibitor carbonyls is observed in most HIV protease
complexes w ith peptidomimetic inhibitors. It was clearly
seen at the difference map though it h as a considerably high
B factor (64 A
˚
2
). High displacement factor is a r esult of
probable disorder of W401 caused b y two orientations of
inhibitor and asymmetrical binding of water with respect to
the noncrystallographic C
2
axis. The positions of two
inhibitor carbonyls (one from Boc group at P
2
position and
another f rom Phe at P
1
¢) that a re bridged b y W 401 are not
symmetrical with respect to the noncrystallographic C
2
axis
and thus the alternative positions of the bridging water
W401 are different. However, only the average position of
W401 was refined in our structure m odel.
In spite o f numerous hydrogen bonds, hydrophobic f orces

seem to be a dominating interaction between protease and
inhibitor. As an indicator of these hydro phobic i nteractions,
distances between carbon atoms o f inhibitor a nd the protease
were calculated using c utoffs of 3.6 A
˚
and 4.1 A
˚
[19]. All
hydrophobic c ontacts and h ydrogen bonds that are involved
in the inhibitor OE binding ar e summarized in Table 3.
Conformation of isostere. Two different conformations of
the isostere of inhibitor fi tting well the electron d ensity were
modeled (Fig. 7). In the first conformation, the NH group
of Phe i n position P
1
¢ makes only one hydr ogen bon d t o one
of t he catalytic aspartates and a lso forms a weak intra-
molecular hydrogen bond to the carbonyl group of the Boc
residue. In the second conformation, the i sostere NH group
binds almost symmetrically between the Asp25 and Asp125
gaining thus additional hydrogen bond to the protease.
However, the
CNS
refinement run with both c onformations
resulted in the same result very similar to the first
conformation. Therefore, the inhibitor was refined in form
that corr esponds to the first c onformation with only one
hydrogen bond to catalytic aspartates not observed with
other inhibitors. However, it seems that the isostere
conformation is probably not fixed and t hat we have to

admit possible concerted confo rmational c hanges at this
site. Because both conformations of the isostere group fit
well into the 2F
o
-F
c
electron density map, we assume that
the inhibitor can change its conformation inside the cavity
and t hat this positively contributes to the i nhibitor binding.
Two fold noncrystallographic symmetry of HIV protease.
The two HIV p rotease subunits are r elated by an approxi-
mate two-fold noncrystallographic axis. Figure 2 shows the
deviations between th e c orresponding ato ms o f chain A and
chain B (averaged f or each re sidue) after the least-squares
Table 3. S ummary of all contacts and hydrogen b onds for inhibitor OE
complexedinthenativeHIV-1protease.Three water molecules
involved in prote in i nhib itor inte raction a re inc lude d. Th e u pper tab le
(six rows) concerns t he I orientation of inhibitor, the lower table ( six
rows) concerns the Y orientation of inhibitor. Contacts to water
molecules (W) are given after the + s ign. Short contacts in th e C-C
column are supposed t o be repulsive, th e short contact s (hydrogen
bonds) in columns 3–5 contribute to good binding a bility of inhibitor
to HIV protease.
Hydrophobic
contacts
Hydrogen
bonds
C-C
up to
4.1 A

˚
C-O/N (+W)
up to
3.6 A
˚
O/N-O/N
up to
3.6 A
˚
O/N-W
up to
3.6 A
˚
I/Boc 14 4 0 2
I/Po0 8 7 + 2 1 1
I/Phe 18 4 + 1 1 1
I/Glu 9 9 6 1
I/Phe+Nh2 10 9 4 2
Total I 59 33 + 3 12 7
Y/Boc 13 1 0 2
Y/Po0 9 7 + 2 1 0
Y/Phe 17 6 + 1 1 1
Y/Glu 10 4 6 1
Y/Phe+Nh2 11 9 4 2
Total Y 60 27 + 3 12 6
Fig. 7. Two conformations of isostere in the inhibitor OE with very
similar energy can be placed in th e 2 F
o
-F
c

electron de n sity o f t he refined
structure of t he complex of H IV-1 protease with OE inhibitor. Torsion
angles of the preferred con formation of the OE isostere a re listed in
Table 4 . O nly one orientation of the disordered inhibitor OE i s shown
in this figure.
Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur. J. Biochem. 271) 4455
alignmentofC
a
atoms. According to expectation, t he main
differences were found in the tips of fl aps where the t wo-fold
noncrystallographic s ymmetry i s not possible b ecause t he
hydrogen bonds connecting tips of flaps cannot be present
with its two-fold image in the same molecule a t once.
Therefore, the alternative positions were modeled at t he
main chains for Ile50-Gly51 and Ile150-Gly151. The least-
squares fit of all C
a
in superimposed monomers of protease
gives th e best over lap b y r otation o f 1 78° with the r msd
0.07 A
˚
for a ll 758 atom pairs and 0.06 A
˚
when the residues
49–53 and 149–153 from the tips of flaps are excluded.
B-factors. The h ighest B factors ( above 50 A
˚
2
) i ndicate high
conformational instability in loops Leu38-Lys44 and

Leu138-Lys144, symmetrically in hinges of both flaps,
whereas the flap ends seem to be well stabilized by
interactions with inhibitor, namely those m ediated by water
molecule locked over the inhibitor carbonyls (Fig. 3). The
map of electron density shows the highest o rientational
disorder in Arg41, Arg141, Lys43 and Lys143 side chains
(zero occupation factors in the PDB fi le).
Adhesion between proteins and protease activity in the
crystal form. Adhe sion b etween p rotein molecules plays
an important role in their function in biological systems
[8]. Structure changes of the protein surface when exposed
to solvent or w hen i nvolved in a dhesion with neighbour-
ing protein molecule have undoubtedly important bio-
logical implications. H ere, the H IV-1 protease molecules
stick together by middle parts of flaps and form a special
helical arrangement with the 6
1
symmetry and with active
sites directed into the wide solvent tunnels passing
through the whole crystal enabling thus an easy exchange
of solvent even in the active sites of proteases. This
explains experimentally verified exchange of inhibitors in
the protease single crystal without an extensive destru ction
of the crystal [20]. In our structure, one of the preferred
interactions between two neighbouring H IV-1 protease
dimers is localized at the top o f flaps. The Phe53 from
one protease dimer and the Phe153 from the other
symmetrically related dimer form a convenient parallel
stacking of phenyl rings joining thus these two dimers
together (Fig. 8A). These p–p interactions appear on both

sides of each p rotease dimer and thus form an infinite
helical arrangement of t he protease complexes i n t he P6
1
space group. This molecular arrangement is supported by
Met46 ( Met146) which forms S…H-C s hort contacts to
Phe53 (Phe153) from the same molecule (Fig. 8B). The
time-averaged view of the protease complex shows that all
residues involved in contact – Phe53, Phe153, Met46,
Met146 were found in two distinct conformations with an
occupation factor of 0.5 (confirmed b y refinement). These
alternative conformations form two favorable parallel
stackings leading to two quite different water channels –
called h ere closed and open solvent channel.
The inner v irtual diameter of the closed solvent channel is
8.7 A
˚
. The inner s urface of the channel is formed by 12 C
c
atoms o f six phenyl pairs (Phe53 and P he153) per one helix
turn (Fig. 9A). The phenyl pairs are h eld together by p–p
interactions and the S
d
atoms of Met46 and Met146 form
close i nteractions with these phenyls. Six of these residue
quartets form a steep spiral ridge i nside the solvent channel.
The virtual diameter of the open solvent channel is
12.4 A
˚
. The surface of the channel is formed by 12 sulfur S
d

atoms of Met46 and M et146 per one turn of helix (Fig. 9B).
In the open solvent channel t he methionines are turned into
the s olvent and do not form significant co ntacts to protein.
Thus, the solvent tunnel cross-section is not rigid because
different c onformations of Phe53, Ph e153, Met46 and
Met146 lead to different tunnel diameters and also to
different hydrophobicity of the s olvent tunnel surface. The
fact that inter–protein interaction can influence the inhibi-
tion process may be important for interpretation of t he
protease function.
Alternative conformations. The H IV protease com plexes
are not rigid. The crystal structure of HIV-1 PR with OE
described here is a mixture o f many conformation states. In
addition to residues Leu38-Lys44 and Leu138-Lys144
localized in flexible flap hinges (see the chapter on
Fig. 8. Dim ers of the complex of HIV-1 protease w ith OE inhibitor are linked together by p–p interactions of phenyl rings o f Phe53 and Phe153 of
neighbouring molecules to f orm a helix along the crystallographic c axis. This p–p interactions are supported by CH…S hydrogen bonds between the
Phe53A … Met46 A and Phe153A … Met146A. (A) Stacking of ne ighbour molecules an d positions of inhibitors in subsequent p rotease dimers
forming th e helix. (B) The detail o f i nteracting residues. P henylalanines a re disordered 1 : 1 in two conformations A and B leading to p arallel
stacking of phenyl rings in each conformation.
4456 H. Petrokova
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2004
B-factors), other eight amino-acid residues h ave been found
each in two distinct alternative conformations: Met46,
Met146, Ile50, Ile150, Gly51, Ile151, Phe53 and Phe153. All
of them have clear interpretation. Two conformations of
Met46 a re associated with corresponding alternative con-
formations of Phe53 so t hat either Met46(A) and Phe53(A)
or Met46(B) and Phe53(B) can be present s imultaneously in

the s tructure and analogously in the second chain o f t he
protease dimer. The residues Met46 and Phe53 placed on
opposite s ides of one flap seem to be of principal importance
for intermolecular arrangement of molecules in c rystal.
Figure 8 s hows t hat the Phe53 rings are in both o rientations
stacked by p–p interaction with Phe153 rings of the
neighbouring protein dimer in crystal. The alternative
positions of phenylalanine rings a re formed in concert with
the alternative positions of S
d
and C
c
of Met46 and Met146
(from neighbouring protease dimer).
Residues Ile50 and Ile150, Gly51 and Gly151 are found
at the tips o f t h e flaps making a h ydrogen bond to each
other. These hydrogen bonds are mediated by NH groups
of one chain with carbonyl groups of the other chain.
Because t he main chains of the t wo loops have the same
orientation of C ends at the place of contact, the
noncrystallographic two-fold s ymmetry cannot be realized
in this location. Therefore, two possible orientations were
modelled in this structure, which differ in t he flipped
peptide bond between residues Ile50-Ile51. The side chains
of neighbouring I le50 and I le150 already fitted well the
electron d ensity in a single position.
Comparison of inhibitors OE, SE and RE complexing
the native HIV-1 protease
The availability of three experimental structure determina-
tions of very similar inhibitors OE, RE and SE complexed

with the native protease gives a detailed i nsight into the
nature of interaction i nhibitor – protease. The chemical
structure o f OE, RE, SE inh ibitors c an be seen from the
scheme in Table 4. T he inhibitors RE, SE differ i n absolute
configuration of the CHOH group of the isostere a nd their
inhibition constants are surprisingly the same K
i
,
SE
¼
0.15 n
M
, K
i
,
RE
¼ 0.12 n
M
[1]. The isostere carboxyl group
replaced in OE by CH
2
makes the inhibition constant
somewhat lower K
i
,
OE
¼ 1.53 n
M
[1]; however , the re sist-
ance of OE to protease mutations seems t o be better.

The experimental structure of HIV-1 protease with
inhibitor SE w as determined by Dohna
´
lek [8] R ¼ 0.18,
diffraction limit 3.1 A
˚
, PDB code 1fqx. The structure with
the RE inhibitor was determined by Dus
ˇ
kova
´
(unpublished
results) with R ¼ 0.173, diffraction limit 2.0 A
˚
.
The overall layout of the inhibitor in the pr otease binding
site is very similar for all the compared inhibitors. The side
chains of all the inhibitors are placed similarly in their
pockets, though, not identically (Fig. 10).
Conformation of inhibitor backbones. The compared
inhibitors differ mainly in their isostere areas. The OE
inhibitor possesses the nonscissile isostere without any
hydroxyl group, whereas the hydroxyethylamine inhibitors
SE an d R E h ave the hydroxyl group of isostere in the S or R
configuration, respectively. The isostere of the OE inhibitor
has quite different confo rmation compared with the R E a nd
SE inhibitors. The inhibitor OE lacks the hydroxyl group
which fixes a uniq ue conformation by strong hydrogen
bonds to Asp25 and/or Gly27 allowing thus in principle
more possible c onformations of the i sostere group. Possible

variations in torsion angles in the inhibitor are cooperative.
This means that any change in o ne torsion angle should b e
compensated by change of other torsion angles in the
opposite direction to keep all the side chains in the whole
Fig. 9. Solv ent t unnels pa ssing through the crystal of HIV-1 protease complexed w ith O E inhibitor along the crystallographic axis c. The residues
Met46, Phe53, Met146 and P he153 participating in the solvent tunnel formation have two different conformations: A, narrow tunnel (A) and B,
wide tunnel (B). The inhibitors OE of six complexes forming one turn o f the helix are also s hown in one orientation in thick stick representation and
coloured ac cording to atom types. I n the case of Ônarrow t unnelÕ, the diameter of t he effective v iew through the solvent ch annel is 8 .7 A
˚
(determined
by distance of projections of two opposite C
c
atoms of Phe53 and Phe153). In the case of Ôwide tunnelÕ, the diameter of t he effective view through the
solvent channel is 12.4 A
˚
(measured a s a distance of projections of opposite S
d
atoms of M et46 and Met146).
Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur. J. Biochem. 271) 4457
inhibitor approximately in the same position. Thus, i n spite
of the fact that the individual torsion angles in the isostere
differ largely (Table 5), the Ôoverall dihedral anglesÕ (i.e. the
dihedral angles determ ining the mutual orientation of the
side chains in S1 and S1¢ sit es) are almost identical (141, 148
and 148° for inhibitors OE, SE and RE, respectively). The
NH group of Phe in P
1
¢ position of the OE inhibitor is
rotated with respect to SE, RE preserving one hydrogen
bond to the catalytic Asp25 only. The i ntramolecular

hydrogen bond NH…O between ester oxygen of Boc and
NH of Phe in P
1
¢ position probably stabilizes this unusual
conformation of the OE inhibitor in the active site. The
aspartate that does not bind to the isostere NH s eems to
make weak hydrogen bonds to both CH
2
groups of the
isostere. The changes of t orsion angles in the isostere also
resulted in shifts of C
a
of Phe i n P
1
¢ position o f t he OE
inhibitor by 0.58 a nd 0.46 A
˚
in comparison with SE and RE
complexes, respectively, in both cases in the direction away
Fig. 10. S tereoview of the position of inhibitor OE (yellow carbons), RE (green carbons) and SE (violet carbons) in the b inding tunnel of native HIV
protease. Conformation of different inhibitors is not identical. Note a different orientation of OH grou ps in isosteres of RE and SE. T h e
S c onfiguration fo rces the OH groups to the inco nvenient orien tat ion f or COH.O h ydrogen b onds with aspartates from one side whereas the
R configuration makes t he same from the opposite side. The c onfo rmation of isostere in the case o f OE seems to be more conformationally flexible.
The p referred c onformation o f OE inhibitor is supported b y the in tramolecular hy drogen b ond CO…HN (P
2
–P
1
¢). Ho wever, mo re conformation
states of OE isostere fit the same m ap of elec tron density ( Fig. 9). Rotatio ns of benzyl groups i n the pro tein pockets P
1

,P
1
¢ and P
3
¢ compensate the
stress imposed by different ge ometries of the H-bond network.
Table 4. T orsion and d ihedral angles (degrees) describing conformation of isostere in inhibitors OE, R E and SE in complex with HIV-1 protease.
Diagram shows s chematically the s tructure of inhbitors and measured torsion a ngles. X ¼ H; (R)OHand(S) O H for OE, R E and SE inhibitors,
respectively.
Inhibitor
Torsion angle Dihedral angle
1.
N
1
-CA
1
-C
1
-C
2
2.
CA
1
-C
1
-C
2
-N
2
3.

C
1
-C
2
-N
2
–CA
2
4.
C
2
-N
2
–CA
2
-C
3
N
1
-CA
1
-C
1
CA
2
-C
3
-N
2
OE 112 -89 165 -21 141

RE 56 -175 -148 77 148
SE 60 -173 -142 67 148
4458 H. Petrokova
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2004
from the catalytic aspartates at the bottom of the active site
tunnel. The shift of inhibitor OE main chain upwards
propagates to P
2
¢ Glu. However, the O
e1
and O
e2
of
inhibitors RE and OE f orm already similar hydrogen bond
pattern to the Asp29 and Asp30 in both cases. T hus, even if
the backbones o f these inhibitors do not follow t he same
line, the h ydrogen bond pattern remains similar, showi ng
high flexibility and adaptation of not only the inhibitor to
the protease but also of the protease to the inhibitor.
Comparison of inhibitors side chains
Boc groups at P
2
of SE and RE inhibitors lie in the same
position. Only in the case of inhibitor OE, the Boc carbonyl
is significantly rotated to form an i ntramolecular hydrogen
bond (with O…N distance 3.6 A
˚
) to NH g roup in P
1

¢
position. This results also in s ignificant s hifts o f t he tert-
butyl group by 0.3 a nd 0.6 A
˚
when compared with SE
and RE inhibitors.
Phenylalanine residues in position P
1
. The orientation of
benzyl group in P
1
position o f the SE inhibitor d iffers from
those in OE and RE. Both, Fig. 10 and T able 6 show that
the rotation of SE benzyl group leads to a lower number of
contacts to protease – compare 12 contacts in SE with 17
and 1 6 contacts in OE and RE, respectively (contact is
defined here as an interatomic d istance lower than 4.1 A
˚
).
Phenylalanine residues in position P
1
¢. The C
a
of OE is
shifted h igher t o t he fl aps than C
a
of SE and RE inhibitors
(about 0.5 A
˚
in both cases). Although phenyl rings of OE

and RE have very similar positions and orientation and
have similar contact patterns (26 and 31 contacts, respect-
ively), the P
1
¢ phenyl ring of SE is turned down to make less
contacts (19) to protease, namely to t he flap r esidues in
comparison with OE and RE inhibitors.
Glutamate residues in position P
2
¢. In comparison with RE
and SE, the C
a
at P
0
2
in OE is shifted upwards to the flaps
and also closer towards the symmetry axis of protease.
However, the Glu Oe
1
and O e
2
in OE and RE overlap and
make the same n etwork of hydrogen bonds to the A sp29
and Asp30 with bo nd-length differences up t o 0.3 A
˚
.Thev
3
torsion angle (C-C-C-OH) of Glu is for all inhibitors
significantly different, namely in t he case of RE (165°, )54°
and )179° for OE, RE and SE, respectively). This results in

different hydrogen bonds between the Glu at P
2
¢ and
protein binding site, although their numbers (6, 7 , 7 , s ee
Table 6 ) remain unchanged.
Phenylalanine residues in position P
3
¢. Benzyl groups of
Phe at P
3
¢ are partially exposed to the solvent in all cases.
However, their positions and orientations are significantly
different for the compared i nhibitors. After the C
a
super-
position, the phenyl rings of different i nhibitors were found
rotatedtoeachother.TheanglesbetweenplainsofP
3
¢
phenyl rings are 98° for OE and SE inhibitors, and 38° for
OE and RE inhibitors. The S3¢ groups of OE and RE
inhibitors having a similar orientation phenyl rings form a
similar number o f close contacts to protease (22 and 20
short C-C contacts for OE, RE, respectively), whereas in the
case of SE inhibitor, there are 13 short C-C contacts to the
protease only (Table 6).
Discussion
The e xperimentally determined structure o f a complex o f
HIV-1 protease w ith O E, RE and SE inhibitors allowed us
to answer the following general questions: (a) why the

presence of hydroxyl group in the i sostere of an inhibitor
(often referred to as necessary replacement of the catalytic
water molecule in r eaction intermediate) is not necessary for
good inhibition properties of inhibitor (b) why the R or S
configuration at t he carboxyl of the isosteric group has no
influence on the inhibition constant, and (c) what is the
influence o f small ch emical changes in the inhibitor molecule
on its conformation in the binding tunnel of HIV protease.
In answer to the first question, it was s hown, that the
hydroxyl binding to catalytic aspartates considered originally
as the m ain condition for good inhibition properties o f
substrate-mimicking inhibitors can be easily replaced by that
of the isostere NH group (if not present as this is the case of
OE). Some e nergy loss c aused by a less dense hydrogen bond
network in place of the missing hydroxyl is probably
Table 5. R eview of interactions of native HIV-1 protease with inhibitors OE, RE, SE ana lysed residue by residue. Comparison of protease residues
which a re in contact with individual si de chains of inhibitors OE , RE and SE. All contacts up to 0.41 nm are accounted for. N umber of contacts t o
certain residue is given.
S
2
S
1
S
1
¢ S
2
¢ S
3
¢
OE RE SE OE RE SE OE RE SE OE RE SE OE RE SE

A28 2 2 – G27 1 3 2 L23 – – 2 I50 – – 2 R8 1 1 1
D29 – – 1 G48 4 1 5 D25 1 1 1 A128 5 2 2 D129 1 1 2
D30 5 6 6 G49 8 5 2 T80 1 1 – D129 3 1 1 G148 13 11 9
V32 2 2 1 I50 2 2 – P81 4 4 1 D130 14 8 11 G149 7 2 1
I47 1 2 4 L123 – 1 – V82 5 5 6 V132 3 2 – F153 – 5 –
G48 2 2 3 P181 1 3 3 I84 7 7 3 I147 5 5 7
I84 2 1 – V182 1 1 – G127 1 – 2 G148 1 – –
I150 2 2 2 G149 2 5 –
I150 7 8 2
16 17 17 17 16 12 26 31 19 31 18 23 22 20 13
Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur. J. Biochem. 271) 4459
compensated by i ncreased flexibility o f the central p art of t he
OE inhibitor leading to a favorable entropy contribution.
In answer to the second questi on, the negligible difference
between inhibition constants o f R E and SE can be explained
by the f act that t he strain imposed on inhibitor during
docking into the binding site of pr otease does not allow
correct orientation of the hydroxyl group to catalytic
aspartates either in S or in the R configuration. The
resulting orientation of the hydroxyl group deviates from
the most convenient direction similarly, but in opposite
directions. In other wo rds, inhibitor remains halfway as far
as correct orientation in both cases is concerned.
In answer to the final question, the X-ray structures
discussed i n this paper showed that some even small changes
in inhibitor chemistry can have a large influence on
conformation of inhibitor main chain. Thus, different
inhibitors differ not only in t he binding affinity, bu t a lso
in the degree o f freedom of the inhibitor inside the binding
tunnel. Some s pecial inhibitors (such a s the OE discussed

here) c an find more conformations in the binding tunnel of
the HIV protease with comparable interaction energies.
This can form a significant contribution to the stability of
the complex through t he entropy term in the average energy
of the system.
Structure determination by X-ray crystallography shows
why t heoretical pred ictions of inhibitor properties have b een
relatively unreliable so far [21]. Even small changes in the
inhibitor chemistry, n o matter w hether they have a high or
small effect on i nhibition constant, result o ften in similarly
small differences in the o verall geometry of th e inhibito r
inside the binding tunnel. Relatively small shifts of C
a
atoms
(about 0.5 A
˚
) and rotations of side chains can s ignificantly
re-form a network of hydrogen bonds, which is, of course,
compensated by a change of the inner torsion energy of
chains destabilizing the co mplex. S eve ral w ater molecules
always taking part in the formation of hydrogen bond
network increase the number of possible configurations and
conformation states of the complex. Calculation of energy
differences between the states containing different number
of atoms and their c onfigurations is a difficult task because
this requires an exact mutual scaling of all the energy
contributions including the entropy and hydrophobic
effects. A good check for selection of a good inhibitor
conformation among many others theoretically possible is
to verify which of them fit at least approximately the

experimental map of electron density that can be nowadays
easily calculated for a ny structure deposited in the P DB
database with its experimentally measured intensities.
Acknowledgements
The work was supp orted by the Grant A gency of the Czech R epublic
(projects 203/98/K023, 204/00/P091, 203/00/D117) and the Grant
Agency of the Academy of Sciences of the Czech Republic (projects
KJB4050312, A4050811, AVOZ4050913). The authors thank the
beamline BM29 staff at ESRF in Grenoble for providing b eam time.
Table 6. C omparison of hydroden bonds (2.4–3.6 A
˚
) of inhibitors OE, RE and SE towards their HIV-1 proteases. Stated numbers are distances
between inhibitor and protease atoms in A
˚
.
Reside of Atoms of Inhibitor complex
Inhibitor Protease Inhibitor Protease OE RE SE
Boc 201 Wat 401 O2 OH2 3.3 3.5 3.5
Po0, Pr0, Ps0 202 Asp 25 OR, OS OD1 2.8
Po0, Pr0, Ps0 202 Asp 25 OR, OS OD2 2.7 3.0
Po0, Pr0, Ps0 202 Asp 125 OR, OS OD2 3.4
Po0, Pr0, Ps0 202 Gly 127 OR, OS O 3.3
Po0, Pr0, Ps0 202 Gly 127 N O 2.7 2.9 3.3
Phe 203 Asp 25 N OD1 3.5
Phe 203 Asp 25 N OD2 3.5 2.8
Phe 203 Asp 125 N OD2 2.5 2.6 3.0
Phe 203 Asp 125 N OD1 3.6 3.3
Phe 203 Wat 401 O OH2 2.4 2.8 2.9
Glu 204 Gly 27 N O 3.2 2.7 3.2
Glu 204 Asp 29 O N 3.0 3.0 2.8

Glu 204 Asp 29 O OD2 3.6 3.4
Glu 204 Asp 29 OE2 N 3.3 3.2
Glu 204 Asp 30 OE1 O 3.5
Glu 204 Asp 30 OE1 OD2 2.8 2.7 3.1
Glu 204 Asp 30 OE1 N 3.0
Glu 204 Asp 30 OE2 OD2 3.6 3.3 3.0
Glu 204 Asp 30 OE2 N 3.1 3.1
Phe 205 Gly 48 N O 2.6 2.9 2.8
Phe 205 Gly 48 O N 3.0 3.0 3.2
Nhh 206 Asp 29 Nhh OD2 3.6 3.6
Nhh 206 Asp 30 Nhh OD2 3.5 3.5
4460 H. Petrokova
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2004
References
1.Babine,R.E.&Bender,S.L.(1997)Molecularrecognitionof
protein-ligand complexes: Applications to drug design. Chem.
Rev. 97, 1359–1472.
2. Leung, D., Abbenante, G. & Fairlie, D. ( 2000) Protease i nhibitors:
current status and fu ture prospects. J. Med. Chem. 43, 305–341.
3. Wlodawer, A . & Eri ckson, J .W. (1993) Structure-based inhibitors
of HIV-1 protease. Annu. Rev. Biochem. 62, 543–585.
4. Berm an, H .M., Battistuz, T., Bhat, T.N., Bluhm, W.F., Bourne,
P.E., B urkhardt, K., Feng, Z ., Gilliland, G.L., I ype, L., Jain, S .,
Fagan, P., Marvin, J., Padilla, D., Ravichandran, V., Schneider,
B., T hanki, N. , Weissig, H ., Wes t brook, J.D. & Zardecki, C.
(2002) The Protein Data Bank. Acta Crystallogr. D58, 899–907.
5. Ko nvalinka, J., Litera, J ., Weber, J., V ond ra
´
s

ˇ
ek, J., Hradilek, M.,
Souc
ˇ
ek, M ., Pichova
´
, I ., Majer, P., S
ˇ
trop, P., Sedla
´
c
ˇ
ek, J ., H euser,
A.M.,Kottler,H.&Kra
¨
usslich, H.G. (1997) Configurations of
diastereomeric h ydroxyeth ylene isosteres strongly affect biological
activities of a series of specific inhibitors of human-immuno-
deficiency-virus proteinase. Eur. J. Biochem. 250, 5 59–566.
6. Rinnova
´
,M.,Hradı
´
lek, M., Bar
ˇ
inka, C., Weber, J., Sou c
ˇ
ek, M .,
Vondra
´

s
ˇ
ek,J.,Klimkait,T.&Konvalinka,J.(2000)Apicomolar
inhibitor of resistant strains of human immunodeficiency virus
protease ide ntified by a c ombinatorial approach. Arch. Biochem.
Biophys. 382, 22–30.
7. Do hna
´
lek, J., Has
ˇ
ek, J., Dus
ˇ
kova
´
, J., Petrokova
´
, H., Hradilek,
M., S ouc
ˇ
ek, M ., Konvalinka, J., Brynda, J., Sedla
´
c
ˇ
ek,J.&Fa
´
bry,
M. (2001) A d istin ct bindin g mod e of a hydroxyethylamine
isostere in hibitor o f HIV-1 prot ease. Acta Crystalogr. D57,472–
476.
8. Do hna

´
lek, J., Has
ˇ
ek, J., Dus
ˇ
kova
´
, J., Petrokova
´
, H., Hradilek,
M., S ouc
ˇ
ek, M ., Konvalinka, J., Brynda, J., Sedla
´
c
ˇ
ek,J.&Fa
´
bry,
M. (2002) H ydroxyethylamine Isostere o f an HIV-1 Protease
Inhibitor Prefers Its A mine to t he Hydroxyl G roup in Bind ing to
Catalytic Aspartates. J. Med. Chem. 45, 1432–1438.
9. Ska
´
lova
´
,T.,Has
ˇ
ek, J., Dohna
´

lek, J., P etrokov a
´
, H., Buchtelova
´
,
E., Dus
ˇ
kova
´
, J ., Souc
ˇ
ek, M., Majer, P., Uhlı
´
kova
´
,T.&Konva-
linka, J. (2003) An Ethylenamine Inhibitor Binds Tightly to Both
Wild Type and Mutant HIV-1 Proteases. Structure and Energy
Study. J. Me d. Chem. 46, 1 636–1644.
10. So uc
ˇ
ek, M. & Urban, J. (1995) An Efficient Method for Pre-
paration of Optically-active N-Protected a-Amino Aldehydes
from N-Protected a-Amino Alkohols. Collect. Czech. Chem.
Commun. 60, 693–696.
11. Dohna
´
lek, J., Has
ˇ
ek,J.,Brynda,J.,Fa

´
bry, M., Sedla
´
c
ˇ
ek, J.,
Konvalinka, J ., Hradilek, M., Souc
ˇ
ek, M ., Adams, M.J. & Nay-
lor, C.E. (1998) Peptidomimetic inhibitors complexed with HIV-I
protease: Crystallization for x-ray diffraction studies. Gen. Physiol.
Biophys. 17, 9–11.
12. Otwinowski, Z. & Minor, W. (1997) Processing o f X-ray D if-
fraction Data Collected in Oscillation Mode. Methods Enzymol.
A276, 307–326.
13. Bru
¨
nger, A.T., Adams, P.D., Clore, G.M., DeLano, W .L., Gros,
P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J ., Nilges, M .,
Pannu,N.S.,Read,R.J.,Rice,L.M.,Simonson,T.&Warren,
G.L. (1998) Cr ystallography & N MR system: a new software suite
for macromolecular structure determination. Acta Crystallogr.
D54, 905–921.
14. Allen, F.H. (2002) The Cambridge Structural D atabase: a quarter
of a million c rystal structures and rising. Acta Crystallogr. B58,
380–388.
15. Jones, T.A., B ergdoll, M. & Kjeldgaard, M. (1990) A macro-
molecular modeling environment. In Crystallographic and Mod-
eling Methods in Molecular Design. (Bu gg, C . & Ealick, S., eds),
pp. 189–195. Springer-Verlag Press, New Y ork.

16. Laskow ski, R.A., M acArthur, M.W., Moss, D.S. & Thornton,
J.M. (1993) Procheck – a program t o check th e stereochemical
quality of p rotein structures. J. Appl. Crystallogr. 26, 283–291.
17. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both
detailed a nd schematic plots of protein structures. J. Appl. Crys-
tallogr. 24 , 946–950.
18. Merritt, E.A. & B acon, D.J. (1997) Raste r3D. P hotorealistic
molecular graphics. Methods Enzymol. 277, 505– 524.
19. Desiraju, G.R. & Steiner, T. (1998) The Weak Hydrogen Bond.
Oxford University Press, New York.
20. Munschi, S., Chen, Z., L i, Y., Olsen, D.B., Fr aley, M.E., Hungate,
R.W. & Kuo, L.C. (1998) Ra pid X-ray diffraction analysi s of
HIV-1 p rotease-inhibito r complexes: inhibitor exch ange in single
crystals of the bound e nzyme. Acta Crystallogr. D54, 1053 –
1060.
21. Garg, R., Gupta, S.P., Gao, H., Babu, M.S., Dabnah, A.K. &
Hansch, C. (1999) Comparative Quantitative Structure-Activity
Relationahip Stu dies on Anti-HIV D rugs. Chem. Rev. 99, 3525–
3601.
Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur. J. Biochem. 271) 4461