Structure of HIV-1 protease in complex with potentinhibitor KNI-272 determined by high-resolution X-rayand neutron crystallographyMotoyasu Adachia, Takashi Ohharaa, Kazuo Kuriharaa, Taro Tamadaa, Eijiro Honjoa, Nobuo Okazakia, Shigeki Araia,Yoshinari Shoyamaa, Kaname Kimurab, Hiroyoshi Matsumurac,d,e, Shigeru Sugiyamac,d, Hiroaki Adachic,d,e,Kazufumi Takanoc,d,e, Yusuke Moric,d,e, Koushi Hidakaf, Tooru Kimuraf, Yoshio Hayashif, Yoshiaki Kisof,and Ryota Kurokia,1

aMolecular Structural Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195,Japan; bDiscovery Research Laboratories, Kirin Pharma Company, 3, Miyahara, Takasaki, Gunma 370-1295, Japan; cGraduate School of Engineering, OsakaUniversity, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; dCore Research for Evolutional Science and Technology, Japan Science and Technology Agency,2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; eSOSHO Inc., 1-6-18 Honmachi, Chuo-ku, Osaka 541-0053, Japan; and fCenter for Frontier Research inMedicinal Science, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan

Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved January 30, 2009 (received for review September 19, 2008)

HIV-1 protease is a dimeric aspartic protease that plays an essentialrole in viral replication. To further understand the catalytic mech-anism and inhibitor recognition of HIV-1 protease, we need todetermine the locations of key hydrogen atoms in the catalyticaspartates Asp-25 and Asp-125. The structure of HIV-1 protease incomplex with transition-state analog KNI-272 was determined bycombined neutron crystallography at 1.9-Å resolution and X-raycrystallography at 1.4-Å resolution. The resulting structural datashow that the catalytic residue Asp-25 is protonated and thatAsp-125 (the catalytic residue from the corresponding diad-relatedmolecule) is deprotonated. The proton on Asp-25 makes a hydro-gen bond with the carbonyl group of the allophenylnorstatine(Apns) group in KNI-272. The deprotonated Asp-125 bonds to thehydroxyl proton of Apns. The results provide direct experimentalevidence for proposed aspects of the catalytic mechanism of HIV-1protease and can therefore contribute substantially to the devel-opment of specific inhibitors for therapeutic application.

drug target � neutron diffraction � reaction mechanism �transition-state analog

The HIV-1 protease (EC 3.4.23.16) is a dimeric asparticprotease that cleaves the nascent polyproteins of HIV-1 and

plays an essential role in viral replication (1–3). Currently, thedevelopment of HIV-1 protease inhibitors is regarded as a majorsuccess of structure-based drug design (4), and the inhibitors ofHIV-1 protease are important compounds for establishing high-ly-active antiretroviral therapy for AIDS (5). Despite this suc-cess, adverse effects linked to the use of HIV-1 proteaseinhibitors and the emergence of HIV-1 mutants resistant toinhibitor action remain critical factors in the clinical failure ofantiviral therapy. The emergence of drug resistance based on therapid rate of viral replication (6) and the high error rate ofreverse transcriptase (7) have become the most urgent concernin HIV-1 treatment.

In considering the goal of effective inhibition of HIV-1protease, the catalytic mechanism must be understood. Thecatalytic mechanism of HIV-1 protease has been inferred fromthe structurally analogous protease pepsin. However, it has beena matter of some debate, with various alternative catalyticmechanisms proposed (8–16). One approach to investigate thecatalytic mechanism of HIV-1 protease is to determine thestructure of a transition-state analog complex, by high-resolutionX-ray and neutron crystallography to identify the location of keyhydrogen atoms. As a transition-state mimetic inhibitor, wechose KNI-272 containing allophenylnorstatine (Apns) withhydroxymethylcarbonyl (HMC) isostere and thioproline (Thp),which is highly selective and potent against HIV-1 protease with

a picomolar inhibitory constant (10, 17, 18). The previous reportof crystal structure showed that the KNI-272 bound to theenzyme exhibited a low energy conformation at an Apns-Thplinkage and 3 water molecules bridged between the inhibitor andenzyme (19). The thermodynamic change upon inhibitor bindingindicates that the process is enthalpy driven, presumably becauseof the burial of a hydrophobic region of the inhibitor; further-more, the contribution of buried water molecules has beensuggested from NMR studies (20, 21).

The HIV-1 protease uses the characteristic aspartyl dyad,which we distinguish by Asp-25 and Asp-125 for consistency withprevious reports (19). Interactions between HIV-1 protease andits inhibitor should strongly depend on the ionization state of thecatalytic active site; indeed, KNI-272 affinity to HIV-1 proteasedepends on pH (21). Although the active-site Asp residues arerelated by a structural dyad in the unbound form, the catalyticmechanism may proceed via asymmetric protonation states.Therefore, determination of the protonation state of the aspartylgroups of enzyme/inhibitor complexes should elucidate theenzymatic mechanism of HIV-1 protease, and subsequentlyprovide knowledge key to improve inhibitor design (22).

To identify the locations of the hydrogen atoms that areimportant for the catalytic action of HIV-1 protease, we usedneutron crystallography. Neutrons strongly interact with hydro-gen and deuterium atoms, and neutron-scattering lengths ofhydrogen and deuterium atoms are very similar to those ofcarbon, nitrogen, and oxygen atoms (23). Detection of hydrogen(deuterium) atoms in water molecules also provides usefulinformation about the dynamic feature of protein-bound watermolecules; there are at least 4 states of protein-bound watermolecules that can be determined by neutron crystallography(24). Neutron diffraction experiments require a relatively largecrystal because of the low flux of neutron beams (106 to 109

neutrons cm�2s�1) (23). We have already succeeded in preparinga large crystal of HIV-1 protease in complex with a transition-state analog inhibitor KNI-272 (25); thus, we performed neutron

Author contributions: R.K. designed research; M.A., E.H., Y.S., H.M., S.S., H.A., K.T., and Y.M.performed research; T.O., K.H., T.K., Y.H., and Y.K. contributed new reagents/analytic tools;M.A., K. Kurihara, T.T., N.O., and S.A. analyzed data; and M.A., K. Kimura, and R.K. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.rcsb.org (PDB ID codes 2ZYE and 3FX5).

1To whom correspondence should be addressed. E-mail: kuroki.ryota@jaea.go.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809400106/DCSupplemental.

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structure analysis of HIV-1 protease in complex with KNI-272 toobserve key hydrogen atoms involved in the catalytic reaction.Here, we show experimental evidence for the protonation statesof the active-site Asp residues of HIV-1 protease obtainedthrough the technique of combining neutron and X-ray crystal-lography using crystals grown under identical conditions andidentical data collection conditions (thus permitting the use ofhigher-resolution phase information with the lower-resolutionneutron-scattering factors).

ResultsStructure of HIV-1 Protease Determined by Neutron Diffraction. Thetertiary structure of HIV-1 protease in complex with inhibitorKNI-272 was determined by a joint X-ray/neutron refinementusing 1.9-Å neutron diffraction data and 1.4-Å X-ray diffractiondata collected at room temperature (Fig. 1). A total of 1,591hydrogen and 520 deuterium atoms and 143 hydration watermolecules were included in the model by comparing the resultsof neutron and X-ray diffraction. Because the data collectionwas performed in a buffer prepared in D2O, 153 side-chainexchangeable hydrogen atoms including catalytic aspartateAsp-25 and KNI-272 were replaced mostly with deuterium atom.The average exchange ratio to deuterium atom was calculated tobe 98.6%. There are also 186 exchangeable hydrogen atoms ofbackbone amides in an asymmetric unit, and average exchangeratio to deuterium atom was calculated to be 53.1% as a resultof occupancy refinement (Table S1). This occupancy value iscomparable with that in the case of dihydrofolate reductase (26).A total of 797 hydrogen bonding interactions including 149hydrogen bonds with hydrated waters were determined.

The overall tertiary structure determined by neutron diffrac-tion at room temperature was closely similar to that of the 0.93-Åresolution X-ray crystal structure determined at 100K: the rmsdfor C� atoms was 0.35 Å.

Interactions Between HIV-1 Protease and KNI-272 at the Active Site.To further confirm the locations of hydrogen and deuteriumatoms in the vicinity of the catalytic residues Asp-25 andAsp-125, the 2Fo � Fc nuclear density map was calculated andcontoured at 1.8 �. The map shows a bulky density near thepositions of the O�2 atom of Asp-25 and O2 atom of KNI-272although the deuterium atom (D�2) from the carboxyl group inAsp-25 and the deuterium atom (DO2) of the hydroxyl group

in the HMC isostere in KNI-272 were omitted (Fig. 2A). TheFo �Fc nuclear density map also shows a strong density belong-ing to deuterium atoms D�2 and DO2 (Fig. 2B). These 2deuterium atoms bind to the carboxyl oxygen (O�2) of Asp-25and the hydroxyl oxygen atom (O2) of the HMC isostere inKNI-272, respectively. The occupancies of hydrogen and deute-rium atoms for both positions were refined to 0.0 and 1.0,respectively. In contrast, no nuclear density for the D�2 atom ofthe carboxyl group in Asp-125 was observed, and no nucleardensity between the 2 catalytic aspartic acids was observeddespite their short distance (3.1 Å) (Fig. 2B).

Fig. 1. Tertiary structure of HIV-1 protease determined by neutron diffrac-tion. The HIV protease dimer is shown by a ball and stick model; watermolecules and bound inhibitor are shown by space-filling representation.Hydrogen and deuterium atoms are colored gray. Carbon (green), oxygen(red), nitrogen (blue), and sulfur (yellow) atoms in protease are indicated.Carbon atoms in KNI-272 are colored dark gray. Figs. 1, 2, and 4 were made byusing the program Pymol (www.pymol.org).

Fig. 2. Neutron and X-ray maps of the active site in HIV-1 protease. (A) 2Fo �Fc nuclear density map contoured at 1.8 �. The deuterium atoms on Asp-25(labeled D�2) and KNI-272 (labeled DO2) are shown, but were omitted for mapcalculation. (B) Fo � Fc omit nuclear density map calculated without thecontribution of the D�2 and DO2 atoms. Maps are shown at 4.5 � (red) and 5.5� (blue) levels. The 2 omitted deuterium atoms are colored cyan. (C) 2Fo � Fc

(cyan) and Fo � Fc (red) electron density maps contoured at 1.5- and 2.5-�values were drawn at 0.93-Å resolution.

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Although no significant additional electron density corre-sponding to these hydrogen atoms could be seen in the 0.93-Åresolution X-ray structure (Fig. 2C), the bond lengths betweenthe carbon (C�) and the 2 oxygen atoms (O�1 or O�2) in Asp-125are nearly equivalent (1.27 and 1.25 Å, respectively), whereas thebond lengths between C� and O�1 or O�2 in the protonatedAsp-25 are 1.20 and 1.32 Å, respectively (Table 1). For com-parison, the bond lengths between C� and O�1 or O�2 in Asp-29and Asp-129, which are responsible for inhibitor binding, arelisted in Table 1. These results show that only the O�2 atom ofAsp-25 is protonated, and Asp-125, Asp-29, and Asp-129 aredeprotonated.

Both side chains of Asp-25 and Asp-125 formed 2 stronghydrogen bonds (�2.7-Å distance) with HMC of KNI-272. Oneis between the D�2 of Asp-25 and the O4 of HMC and the otheris between DO2 of hydroxyl group in HMC and O�1 of carboxylgroup in deprotonated Asp-125. The distances between thedeuterium and acceptor atoms are relatively shorter (1.8–1.9 Å)than other hydrogen bonds with KNI-272 (Table 2).

Water Molecules Localized at the Interface Between KNI-272 and HIV-1Protease. There are 6 water molecules (HOH301, HOH322,HOH354, HOH566, HOH607, and HOH608) within a distance

of 3.5 Å from KNI-272 as shown in Fig. 3 and Table S2).Deuterium atoms of these water molecules were confirmed inthe 2Fo � Fc and Fo �Fc omit maps. Three water molecules(HOH301, HOH566, and HOH608) mediating hydrogen-bonding interactions between HIV-1 protease and KNI-272show clear nuclear densities for deuterium as shown in Fig. 4.The water molecule, HOH301, located at the symmetric positionof the dimer interface forms hydrogen bonding interactions withKNI-272 and the main chain nitrogen (N) atoms of Ile-50 andIle-150 in the ‘‘f lap’’ structure of HIV-1 protease (Fig. 4A).HOH607 participates as a proton donor in hydrogen bondinginteractions with Gly-27 and the catalytic residue Asp-125.Collectively, a total of 16 hydrogen bonds were identified in theinteraction between HIV-1 protease and KNI-272, and 9 of 16hydrogen bonding interactions were water mediated.

Table 1. Bond distances between carbon and oxygen atoms inthe carbonyl group of Asp residues responsible for interactionwith KNI-272

ResidueCarbonatom

Oxygenatom

Distance,Å

EstimatedSD, Å

Asp-25 C� O�1 1.203 0.007Asp-25 C� O�2 1.322 0.007Asp-29 C� O�1 1.242 0.008Asp-29 C� O�2 1.261 0.009Asp-125 C� O�1 1.265 0.008Asp-125 C� O�2 1.251 0.008Asp-129 C� O�1 1.244 0.008Asp-129 C� O�2 1.250 0.008

The bond distances were calculated by using the structure determined by0.93-Å resolution X-ray crystallography.

Table 2. Direct hydrogen bonds and water molecule-mediated hydrogen bonds between HIV-1 protease and KNI-272 determined byneutron crystallography

Donoratom

Deuteriumatom

Acceptoratom Distance*, Å Distance†, Å Angle‡, °

Asp-25 O�2 Asp-25 D�2 KNI O2 3.0[3.1] 2.3 124Asp-25 O�2 Asp-25 D�2 KNI O4 2.7[2.6] 1.9 144Asp-29 N Asp-29 D HOH608 O 3.0[2.9] 2.1 160Ile-50 N Ile-50 D HOH301 O 3.1[3.0] 2.3 157Asp-129 N Asp-129 D KNI O3 2.9[2.9] 2.0 158Ile-150 N Ile-150 D HOH301 O 3.1[2.9] 2.2 165KNI N2 KNI DN2 Gly-148 O 2.9[2.8] 2.2 131KNI N3 KNI DN3 Gly-127 O 3.1[3.1] 2.2 156KNI N5 KNI DN5 HOH608 O 3.1[3.1] 2.1 167KNI O2 KNI DO2 Asp-125 O�1 2.7[2.6] 1.8 154KNI O2 KNI DO2 Asp-125 O�2 3.2[3.2] 2.4 144HOH301 O HOH301 D1 KNI O6 2.8[2.9] 1.9 146HOH301 O HOH301 D2 KNI O5 2.8[2.8] 1.8 168HOH566 O HOH566 D1 Asp-129 O�1 3.0[2.9] 2.0 163HOH566 O HOH566 D2 Gly-127 O 2.8[2.8] 2.1 128HOH566 O HOH566 D2 KNI O3 3.2[3.0] 2.6 117

The numbers in square brackets indicate the value obtained from X-ray structure at 100 K.*Distance between donor and acceptor atoms.†Distance between deuterium and acceptor atoms.‡Angles of donor–deuterium–acceptor atoms.

Fig. 3. Schematic diagram of the interaction between HIV-1 protease andKNI-272 (bold lines). Hydrogen bonds are shown by broken lines. Asterisksindicate the hydrogen atoms replaced with deuterium atom (occupancies ofdeuterium atom are �0.5).

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DiscussionKNI-272 is a unique inhibitor designed as a transition-stateanalog for peptide bond hydrolysis by HIV-1 protease (17, 18).Because the locations of the hydrogen atoms of catalytic aspar-tates in HIV-1 protease have not previously been experimentallydetermined, neutron crystallography of HIV-1 protease in com-plex with KNI-272 was performed to permit elucidation of theenzymatic mechanism of HIV-1 protease.

The neutron structure determination of HIV-1 protease dem-onstrated that the carboxyl group of Asp-25 is protonated, whereasthat of Asp-125 is deprotonated. In previous reports, the ionizationstates of the catalytic residues of HIV-1 protease in complex ofKNI-272 or pepstatin A were investigated by pD(or pH) dependence of chemical shift and the H/D isotope effectby using 13C NMR (27, 28). Although the chemical shift of bothAsp-25 and Asp-125 had not been changed through pD 2.5–6.2, theresults of the isotope effect indicated protonation of Asp-25. Incontrast, ab initio molecular dynamics-based assignment suggestedthat both aspartic groups are protonated, and a hydrogen bond isformed between the 2 aspartic acids (29). When considering these2 alternatives, the neutron diffraction data support protonation ofAsp-25 and the isotope effect results.

It has been suggested that a low-barrier hydrogen bondinginteraction, a unique hydrogen bond with 2 acceptors sharing ahydrogen atom (30, 31), occurs between the carboxyl groups ofAsp-25 and Asp-125 in the absence of bound inhibitor (11) andin product complex (14). However, we could not detect anysignificant nuclear density between Asp-25 and Asp-125 in ouranalysis, probably because the side-chain conformations of thesecatalytic aspartates are fixed by hydrogen-bonding interactionwith the main-chain N atoms of Gly-27 and Gly-127.

The catalytic mechanism of HIV-1 protease has been exten-sively investigated by several approaches (12). Our determina-tion of the protonation state and location of deuterium atoms inthe enzyme/inhibitor complex will help further understanding ofthe enzymatic mechanism of HIV-1 protease. As shown in Fig.5, we summarize a possible model for the catalytic mechanism ofHIV-1 protease consistent with our current neutron diffractiondata and previous literature (8, 10, 15, 32, 33). The structure ofthe HIV-1 protease/KNI-272 complex displays the structuralcharacteristics of a tetrahedral transition-state complex (state *1in Fig. 5). Whereas the hydrogen bond between the carbonylgroup in HMC and Asp-25 appears to be the primary interactionwith substrate, the location of the hydroxyl group in HMCappears ideal to mimic the location of an attacking watermolecule in catalysis. Taken together, our results demonstratethat Asp-25 provides a proton to the carbonyl group of thesubstrate and Asp-125 contributes to activate the attacking watermolecule as a nucleophile. Because the inhibitor does not allowhydrolysis to proceed, it is concluded that the protonation statesof both the catalytic residues in the KNI-272 complex retainsthose within the ES complex.

Fig. 4. Neutron and X-ray maps of water molecules involved in the water-mediated hydrogen bonds between HIV-1 protease and KNI-272. Shown arethe structural environment of HOH301 (A), HOH566 (B), and HOH608 (C).2Fo � Fc X-ray maps drawn in blue were contoured at 3.0 �. Fo � Fc nucleardensity maps drawn in red were calculated after omitting D1 or D2 atoms ofwater molecules and are contoured at the 3.0-� level. Hydrogen bonds arerepresented by dotted lines in pink.

Fig. 5. A proposed energy profile for the catalysis by HIV-1 protease. E, P, and S denote enzyme, products, and substrate, respectively. The reaction coordinateof the separated states is referred from the results of Bjelic and Aquvist (15).

4644 � www.pnas.org�cgi�doi�10.1073�pnas.0809400106 Adachi et al.

There are also several reports describing the X-ray structure ofHIV-1 protease in complex with not only product (14, 34, 35) butalso a tetrahedral intermediate (16, 33, 36). It appears that the 2oxygen atoms of hydroxyl (O2) and carbonyl oxygen (O4) in HMCof KNI-272 are located at similar positions to the oxygen atoms inthe gem-diol of hydrated substrate (16) and hydrated KVS-1inhibitor (33). However, Kumar et al. (36) reported an importantobservation of the interaction between the catalytic residues andthe intermediate. They observed hydrogen bond formation be-tween one catalytic residue and nitrogen atom of the intermediate,which is distinct from our observation; and each observation maysuggest a different step in catalytic reaction.

Although current inhibitor design is focused on improvementsin affinity and specificity for target proteins, the inhibitor designfor HIV-1 protease is more focused on its drug resistance; oneof the efficient approaches is to improve interaction with thestructurally-essential regions in HIV-1 protease, such as catalyticresidues or key regions of the protein backbone. According tocalorimetric binding experiments performed as a function of pH,efficient KNI-272 binding requires protonation of a catalyticresidue because the affinity of KNI-272 decreases above pH 6(21) (likely caused by the deprotonation of Asp-25 from ourneutron data). This insight leads us to hypothesize that anefficient inhibitor would retain key interactions seen in KNI-272binding and neutralize the charged state of the catalytic residues.Consequently, introduction of a positive charge in an inhibitor atthe active site would be favorable because of the charge–chargeinteraction with a deprotonated catalytic residue isolated fromsolvent. With regard to the use of backbone interactions with aninhibitor, there is an example of darunavir containing bis-THFforming hydrogen-bonding interactions with main-chain atomsof Asp-29 and Asp-30, which might account for potent inhibitionagainst highly drug-resistant mutants of HIV-1 protease (37).

Displacement of a water molecule interacting with an inhibitormight lead to increased affinity to HIV-1 protease mutants froman entropic point of view. Six water molecules (HOH301,HOH322, HOH354, HOH566, HOH607, and HOH608) directlybound to KNI-272 may be candidates for displacement (Table S2and Fig. 3). Indeed, displacing a water molecule correspondingto HOH301 was used for designing cyclic urea-based inhibitors(38). The results of NMR (water/NOESY and water/rotating-frame Overhauser effect spectroscopy) showed that HOH301,HOH566, and HOH608 are long-lived water molecules respon-sible for binding of KNI-272 to HIV-1 protease (20). It wasreported that the favorable binding enthalpy is caused by inter-actions with long-lived water molecules around KNI-272 (21).Molecular dynamics simulation indicated that HOH301 andHOH607 significantly contribute to the binding free energy (39).These water molecules have relatively small B factors in thestructures determined by 1.9-Å neutron and 0.93-Å X-ray struc-ture analyses. Overall, therefore, the structural analyses includ-ing protonation status of the catalytic residues and the infor-mation for the bridging water molecules determined in this studyprovide important information applicable to the design ofpotentially novel and specific HIV-protease inhibitors.

MethodsPreparation of HIV-1 Protease. Preparation of HIV-1 protease was performedas reported (25). In brief, the chemically-synthesized DNA encoded the genefor the initial methionine and the 99-aa HIV-1 protease including 5 mutationsof Q7K, L33I, L63I, C67A, and C95A (to prevent autoproteolysis and cysteinethiol oxidation) (40) were used for expression in Escherichia. coli. The ex-pressed protein was refolded and purified by cation exchange chromatogra-phy followed by reversed-phase chromatography. The final yield of HIVprotease was �30 mg from 6-L cultivation determined by using UV absorptionat 280 nm with a molecular extinction coefficient of 8,600 cm�1M�1.

Crystallization of HIV-1 Protease. Crystallization and crystal growth wereperformed as reported (25). Briefly, a crystal of HIV-1 protease complexed with

KNI-272 (�0.3 � 0.3 � 0.1 mm) was grown and used as a seed to grow a largercrystal suitable for neutron diffraction studies. Approximately 2 mg of proteinsolution in 0.125 M citrate/0.25 M phosphate buffer (pH 5.5) was used for seedcrystal growth; this crystal was then transferred to a protein solution contain-ing 1.5 mg/mL of HIV-1 protease. A large crystal was subsequently obtainedwith the dimension of 3.6 � 2.0 � 0.5 mm. For neutron diffraction datacollection, the crystal was soaked for 2 weeks at 293 K in the crystallizationsolution (pD 5.0), containing D2O. This pD 5.0 is near the optimum in enzymaticactivity of HIV-1 protease (11). A crystal obtained at the same crystallizationcondition was used for X-ray diffraction data collection at room temperature.Before data collection, the crystal was also soaked into D2O based crystalliza-tion solution (pD 5.0) at 293 K.

Data Collection. Neutron diffraction data were collected to 1.9-Å resolution byusing a crystal (3.6 � 2.0 � 0.5 mm) at room temperature on the BIX-4diffractometer (41) installed at the 1G-A site of the JRR-3 reactor in the JapanAtomic Research Agency. Data collection was carried out by using the step-scan method with an interval angle of 0.3° and exposure times of 360 min perframe. The total time required to collect the total of 181 frames was 46 days.The diffraction data were integrated, scaled, and merged by using the pro-grams DENZO and SCALEPACK (42). The detailed statistics of data collectionare listed in Table S3. Additional X-ray diffraction data were collected to 1.4-Åresolution at room temperature for the use of joint refinement. The datacollection was performed by using the oscillation method with rotation angleof 1° and exposure times of 5 s per frame at BL6A at the Photon Factory(Ibaraki, Japan). The diffraction data were integrated, scaled, and merged byusing the program HKL2000 (42).

Ultra high-resolution X-ray diffraction data were collected to 0.93 Å with acrystal (0.6 � 0.2 � 0.1 mm) at 100 K at the BL41XU beamline at SPring-8(Hyogo, Japan). The crystal was soaked into the precipitant solution contain-ing 45% (wt/vol) glycerol, then flash-frozen under a N2 gas cryostream (100 K).The high-resolution diffraction dataset containing 180 frames was collectedwith exposure times of 10 s per frame and by changing the X-ray beamposition along the crystal every 36 frames. Subsequently, a dataset to accu-rately collect the lower-resolution diffraction data was collected with anexposure time of 1 s per frame. The diffraction data containing a total of 360frames were integrated, scaled, and merged by using the programs HKL2000(42). The detailed statistics of data collection are listed in Table S3.

Structure Determination. Refinement of the 0.93-Å-resolution X-ray structurewas carried out by using the program CNS (43) followed by SHELX-97 (44) withmanual model adjustment using XtalView (45). X-ray structure [Protein DataBank ID code 1HPX (19)] was used as the starting model for refinement. Eachcrystallographic asymmetric unit contained a HIV-1 protease dimer comprising2 equivalent monomers residues distinguished by the residue numbers 1–99and 101–199. Although HIV-1 protease was expressed with an extra Met at theN terminus, any electron density maps of the Met residue were not observed.Hydrogen atoms were included in the model by SHELXL-97 as riding hydro-gens. Restraints (DFIX and DANG) of carboxyl atoms in Asp residues werefinally released. SHELX-97 was used to calculate the estimated standarddeviation for carboxyl bond length. The final model contained alternativeconformations of 36 amino acid residues. Refinement statistics are also givenin Table S3.

A coordinate set for neutron structure was uniquely obtained by using ajoint refinement method (46) with the computer program PHENIX (47) and the1.9-Å neutron and 1.4-Å X-ray diffraction datasets collected at room temper-ature with a crystal prepared under identical conditions. X-ray structuredetermined at 0.93-Å resolution was used as a starting model of refinement.The hydrogen atoms in the protein and water were initially placed by usingprograms PHENIX (47) and CNS (43). These locations were manually adjustedwith the program XtalView (45). Solvent-accessible and -exchangeable hydro-gen atoms in side chains of Arg, Asn, Gln, His, Lys, Ser, Thr, Trp, and Tyr werereplaced with deuterium atoms. Both hydrogen and deuterium atoms wereadded to the buried side chains (Thr-26, Thr-31, and Thr-80) and the side chainsof catalytic residues and KNI-272 at exchangeable sites, and the occupanciesof deuterium/hydrogen atoms were subsequently evaluated. There are 3asparagines and 5 glutamines in the HIV-1 protease structure; the side-chainamide conformations for Gln-2, Gln-18, Gln-58, Gln-102, and Asn-198 werecorrected based on the nuclear density map from neutron crystallography.D2O molecules were placed according to the locations of oxygen atomsdetermined by using the 1.4-Å-resolution X-ray data, and then deuteriumatoms were placed into the positive densities observed in neutron 2Fo � Fc

maps. Finally, 143 water molecules were included for the joint refinement byusing neutron and X-ray diffraction data. The R and Rfree values for the finalmodel were 19.3% and 22.2%, respectively, as summarized in Table S3.

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ACKNOWLEDGMENTS. We thank the beamline staff at the SPring-8 (Drs. N.Shimizu and M. Kawamoto) and the Photon Factory (Profs. N. Igarashi and S.Wakatsuki) for help and Prof. M. Blaber for critical reading of this manuscript.The synchrotron radiation experiments were performed at the BL41XU beam-line in SPring-8 with the approval of the Japan Synchrotron Radiation Re-

search Institute (Proposal 2007A1513) and at the BL6A beamline at the PhotonFactory (Proposal 2007G212). This work was supported in part by the Ministryof Education, Culture, Sports, Science, and Technology of Japan, Grant-in-Aidfor Young Scientists B17710190 (to M.A.), and Grant-in-Aid for ScientificResearch B19370046 (to R. K.).

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