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

Weber1,5 1 Department of Biology, Georgia State University, Atlanta, GA, USA;2Biochemistry and Molecular Biology Department, University of Debrecen, Hungary;3Laboratory of Chemical Physi

Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site

Bhuvaneshwari Mahalingam1,*, Yuan-Fang Wang1, Peter I Boross1,2, Jozsef Tozser2, John M Louis3, Robert W Harrison1,4and Irene T Weber1,5

1

Department of Biology, Georgia State University, Atlanta, GA, USA;2Biochemistry and Molecular Biology Department, University

of Debrecen, Hungary;3Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, The National Institutes of Health, Bethesda, MD, USA;4Department of Computer Science, Georgia State University,

Atlanta, GA, USA,5Department of Chemistry, Georgia State University, Atlanta, GA, USA

The crystal structures of the wild-type HIV-1 protease (PR)

and the two resistant variants, PRV82Aand PRL90M, hav e

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 (Ki) of PRV82A and PRL90Mwas 3.3- and

0.16-fold, respectively, relative to the value for PR They showed

only a modest decrease, of 10–15%, in their kcat/Kmvalues

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 PRV82Ashowed local changes

in residues 81–82 at the site of the mutation, while PRL90M 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 infectivspecific-ity 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¢, which span 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: iweber@gsu.edu

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)

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, PRV82Aand PRL90M, 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 PRV82A

and PRL90M 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

37C 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 nM, was added to varying concentrations of

sub-strate (25–400 lM), maintained in 50 mM sodium acetate,

pH 5.0, containing 0.1M NaCl, 1 mM EDTA, 1 mM

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 usingSIGMAPLOT8.0.2 (SPSS Inc., Chicago, IL, USA)

The Ki values were obtained from the 50% inhibitory

concentration (IC50) values, estimated from an inhibitor

dose)response curve with the spectroscopic assay and using

the following equation:

Ki¼ ½ðIC50 ½E=2Þ=ð1 þ ½S=KmÞ

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 0–4.0M urea, using the spectroscopic assay Initial velocities were plotted against urea concentration and fitted to a curve for solvent denaturation usingSIGMAPLOT8.0.2

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 PRV82A crystals were obtained using 0.05M citrate/phosphate buffer, pH 5.0– 5.6, with 0.2–0.4MNaCl as precipitant, while the PRL90M crystals were obtained using 0.1Msodium acetate buffer,

pH 4.6, with 2M 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 (PRL90M) and 1SDV (PRV82A) 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, PRV82Aand PRL90M, 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 kcatvalue compared to PRV82Aand PRL90M, respectively The kcat/Km values of PRV82A and PRL90M were  85% and 81%, respectively, relative to PR The PRL90Mhydrolysis of three other peptide substrates has shown kcat/Km values of

 40%, relative to PR, albeit under different assay condi-tions [12] The Ki values for the three enzymes showed a greater variation than the k /K values The apparent

binding affinity of indinavir to PRV82Awas about three-fold

lower than that of PR Klabe et al [28], using different assay

conditions, reported a six-fold lower Ki value for the

PRV82A–indinavir interaction relative to that observed with

PR The six-fold stronger inhibition of PRL90Mby 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 PRV82A and

PRL90M, respectively These DDG values correspond to the

loss in PRV82A, or gain in PRL90M, of about one hydrogen

bond or van der Waals contact relative to the PR interaction

with indinavir

The stability of PRV82A, as assessed by urea denaturation,

was similar to that of PR, whereas PRL90Mwas significantly

more sensitive to urea In general, PRV82A, which alters a

residue in the inhibitor-binding site, showed similar activity

and stability, and lower inhibition, relative to PR However,

the nonactive site mutant, PRL90M, 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, PRV82A and

PRL90M 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 P21212 included two chloride ions, while the

L90M structure in space group P212121included 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>PRL90M>

PRV82A 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 PRL90M

(Fig 2) In PR , 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

PRL90M 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)1Æl M )1 )

K i (p M )

DDG (kcalÆmol)1)

UC 50 ( M )

a Taken from Mahalingam et al [12].

Table 2 Crystallographic data statistics RMS, root mean square.

Protease mutant

Space group P2 1 2 1 2 P2 1 2 1 2 P2 1 2 1 2 1 Unit cell dimensions (A˚)

Overall (final shell) (25.8) (19.6) (37.4)

Overall (final shell) (7.96) (8.17) (4.41) Resolution range for

refinement (A˚)

Overall (final shell) (87.8) (91.5) (78.0) RMS deviation from ideality

Average B-factors (A˚2)

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 PRV82A, and both the Od

atoms of Asp25¢ in PRL90M, 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 PRL90Mand 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

The side chains of Met90 and Met90¢ in the PRL90M 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

PRV82A 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

PRL90M 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 PRL90M(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

residues of PR (- - -) and PR L90M (––) The residues in the two subunits

are numbered 1–99 and 1¢ )99¢ The B-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

Alternate conformation

Disordered density

Met46 Met46¢ Met46 Met46¢, Met46

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 PRV82A showed 95 van der Waals contacts with indinavir, similar to the PR However,

PRL90M 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) Comparison of Met90¢ 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.

Arg8 a

Arg8¢ b

Gly27 a

Gly27¢ a

Asp29¢ a

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.

actions, including the same water-mediated interactions,

except for one new interaction in PRL90M (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 PRV82A However, PRL90M 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 PRL90M, the

pyridyl N5 of indinavir formed a hydrogen bond with

the NH of Arg8¢ (3.1 A˚) The corresponding distances in

PR and PRV82Awere 4.0 and 4.2 A˚, respectively This new interaction could explain the better inhibition of PRL90M

by indinavir

Structural differences between mutants and PR The PRV82Astructure 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 PRV82A, 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

PRV82A, 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 kcat/Km and the three-fold increased Kifor indinavir with PRV82A relative to PR

PRL90Mshowed 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 PRL90M have

Table 5 Protease–indinavir hydrogen bond interactions.

Indinavir

Water Protease PR PR V82A PR L90M 1HSG

Direct interactions

Water-mediated interactions

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).

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 PRL90Mhave

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

PRL90M, 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 PRL90Mcompared 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 PRL90M, relative to their positions in PR and

PRV82A 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 PRL90Mrelative

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 DDG of)1.19 kcal/mol for the inhibition of PRL90Mcompared to PR

The structural changes described for PRV82A and

PRL90M, 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 PRV82A and PRL90M showed opposite changes in the main chain atoms of residues 81–82, and PRL90Malso 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, PRV82Aand PRL90M, were compared by using crystallographic and kinetic analysis The two mutants showed slightly decreased kcat/Kmvalues as compared to

PR PRV82Aand PRL90M had increased and decreased Ki 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,

PRV82A, 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, PRL90Mshowed 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 Ki of PRL90M 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 PRL90Mis 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 PRV82Aof about one van der Waals contact and gain in PRL90Mof 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.

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.

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