Proc. Natl. Acad. Sci. USAVol. 92, pp. 6057-6061, June 1995Biochemistry

Catalytic domain of human immunodeficiency virus type 1integrase: Identification of a soluble mutant by systematicreplacement of hydrophobic residuesTIMOTHY M. JENKINS, ALISON B. HICKMAN, FRED DYDA, RODOLFO GHIRLANDO, DAVID R. DAVIES,AND ROBERT CRAIGIE*Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892-0560

Communicated by Kiyoshi Mizuuchi, National Institutes of Health, Bethesda, MD, March 24, 1995

ABSTRACT The integrase protein of human immunode-ficiency virus type 1 is necessary for the stable integration ofthe viral genome into host DNA. Integrase catalyzes the 3'processing of the linear viral DNA and the subsequent DNAstrand transfer reaction that inserts the viral DNA ends intohost DNA. Although full-length integrase is required for 3'processing and DNA strand transfer activities in vitro, thecentral core domain of integrase is sufficient to catalyze anapparent reversal ofthe DNA strand transfer reaction, termeddisintegration. This catalytic core domain, as well as thefull-length integrase, has been refractory to structural studiesby x-ray crystallography or NMR because of its low solubilityand propensity to aggregate. In an attempt to improve proteinsolubility, we used site-directed mutagenesis to replace hy-drophobic residues within the core domain with either alanineor lysine. The single substitution of lysine for phenylalanine atposition 185 resulted in a core domain that was highly soluble,monodisperse in solution, and retained catalytic activity. Thisamino acid change has enabled the catalytic domain ofintegrase to be crystallized and the structure has been solvedto 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T. M.,Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266,1981-1986]. Systematic replacement of hydrophobic residuesmay be a useful strategy to improve the solubility of otherproteins to facilitate structural and biochemical studies.

Retroviral replication requires the integration of a lineardouble-stranded DNA copy of the viral RNA genome into ahost cell chromosome (1, 2). Prior to integration, cleavage ofthe 3' ends of the linear viral DNA removes two nucleotides3' of a conserved CA dinucleotide. The processed 3' ends ofthe viral DNA are then covalently joined to host DNA in asubsequent DNA strand transfer reaction. In the resultingintegration intermediate (3, 4), the 5' ends of the viral DNAand the 3' ends of the host DNA at the site of integrationremain unjoined. Host enzymes are probably responsible forrepairing the single-strand connections between viral and hostDNA to complete the integration process. Regions of the viralgenome that are essential for integration map to the 3' end ofthe pol gene, which encodes the integrase (IN) protein (5-8),and to the ends of the viral DNA (9-12).

Purified recombinant human immunodeficiency virus type 1(HIV-1) IN performs both 3' processing and DNA strandtransfer reactions in vitro with oligonucleotide substrates thatmimic the ends of HIV-1 DNA (13-15). IN can also catalyzean apparent reversal of the strand transfer reaction, termeddisintegration (16). Deletion studies on HIV-1 and HIV-2 INhave revealed that although both the 3' processing and DNAstrand transfer reactions require a full-length protein (17, 18),the central core domain is able to catalyze the disintegration

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

reaction (19). This catalytic domain, which is relatively resis-tant to proteolysis (20), contains the conserved residues AspM6,Asp116, and Glu152, the D,D-35-E motif found in all retroviralintegrases. These acidic residues, which also form the samemotif in all retrotransposons and certain bacterial transposableelements, may coordinate metal ions directly involved incatalysis (20-23).The propensity of IN to aggregate at concentrations below

those required for many physical methods has frustratedstructural and biophysical studies. In particular, aggregationhas prevented structural analysis by x-ray crystallography orNMR. We report that a single amino acid substitution, Phe185-- Lys (F185K), in the catalytic core domain of HIV-1 IN resultsin a protein that is dramatically more soluble, is monodisperse insolution, and retains catalytic activity. The mutation has enabledthis domain to be crystallized and the structure has been solvedto 2.5-A resolution (24).

MATERIALS AND METHODSConstruction ofSite-Directed Mutant Plasmids. General tech-

niques for manipulating DNA were as described (25). DNAfragments encoding site-directed mutations were generated by an"overlapping" PCR protocol (26), using Vent DNA polymerase(New England Biolabs) according to the manufacturer's guide-lines. Primers were designed to generate DNA fragments con-taining an Nde I site at the 5' termini and a stop codon flankedby a BamHI site at the 3' termini. These PCR fragments wereproduced in a two-step procedure. In a first round of PCR, DNAfragments were generated by using plasmid pINSD (20) astemplate DNA. Amplification using the 5' (Nde I site) primerwith a 3' primer containing a mutation produced a PCR "half-fragment." Separate amplification with the 3' (BamHI site)primer and the 5' primer containing the mutation produced theother half-fragment. In a second round of PCR, the two over-lapping half-fragments, after gel purification, were mixed to-gether with the two external restriction site-containing primersfor a final PCR to generate a full-length DNA fragment con-taining the desired mutation. After gel purification and cleavagewith BamHI and Nde I, the full-length DNA fragments wereligated into pET-15b (Novagen), placing the site-directed mu-tants under the control of a T7 promoter (27). This vectorencodes a 20-aa histidine tag (HT) at the amino terminus(MGSSHHHHHHSSGLVPRGSH-) that allows rapid purifica-tion of the expressed protein on a nickel-chelating column. Thissequence also contains a thrombin cleavage site (LVPRGS) toallow removal of the HT after purification.

Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; HIV-1, human immunode-ficiency virus type 1; HT, histidine tag; IN, integrase.*To whom reprint requests should be addressed at: Building 5, Room301, Laboratory of Molecular Biology, National Institute of Diabetesand Digestive and Kidney Diseases, National Institutes of Health,Bethesda, MD 20892; electronic mail-mail:bobC@helix.nih.gov.

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Protein Expression and Solubility Measurements. Plasmidsencoding the site-directed mutants were expressed in Esche-richia coli BL21(DE3) (28). Protein expression was induced asdescribed (13), with slight modifications (20).The solubility of each HT-IN-(50-212) mutant protein was

examined in crude cell lysates. Cells harvested from 1 liter ofSuper broth (Biofluids) containing 100 ,ug of ampicillin per mlwere resuspended in 12 ml of 25 mM Hepes (pH 7.5) andfrozen in liquid N2. Resuspended cells (100 ,ul) were lysed bythe addition of NaCl (0.15 M, 0.5 M, or 1 M), dithiothreitol(DTT, 2 mM), and lyzozyme (0.3 mg/ml), in a final volume of170 ,ul. Cells were incubated 30 min on ice, frozen in liquid N2,and thawed. The lysate was centrifuged in a Beckman TL-100ultracentrifuge for 45 min at 100,000 x g and the supernatantwas recovered. Ten microliters of supernatant was mixed with2 ,ul of protein sample loading buffer [0.36 M Tris HCl, pH6.8/10% (wt/vol) SDS/40% (wt/vol) glycerol/50 mM DTT/0.005% (wt/vol) bromophenol blue], heated at 100°C for 5min, and analyzed by SDS/PAGE. Protein was detected bystaining with Coomassie blue R-250.

Protein Purification and Disintegration Activity. Cells ex-pressing the mutant that contains the single substitution oflysine for phenylalanine at position 185 [HT-IN-(50-212/F185K)] were harvested from a 1 liter culture and suspendedin 12 ml of 25 mM Hepes (pH 7.5) before freezing in liquid N2.Thawed cells were suspended in lysis buffer (25 mM Hepes, pH7.5/0.5 M NaCl/2 mM 2-mercaptoethanol/5 mM imidazolewith lysozyme at 0.3 mg/ml) to a final volume of 40 ml. Lysedcells were sonicated and then centrifuged at 30,000 x g for 40min. The supernatant was filtered through a 0.45-,um filter andapplied to a Ni-affinity (chelating Sepharose; Pharmacia)column (0.5 x 8 cm). After extensive washing with 20mM and60 mM imidazole in elution buffer [25 mM Hepes, pH 7.5/0.5M NaCl/2 mM 2-mercaptoethanol/10% (wt/vol) glycerol],protein was eluted with a 10-column-volume linear gradient of60 mM to 1 M imidazole in elution buffer. Pooled fractionscontaining HT-IN-(50-212/F185K) were dialyzed against 25mM Hepes, pH 7.5/0.5 M NaCl/1 mM DTT/1 mM EDTA/10% (wt/vol) glycerol. The HT was removed by cleavage withthrombin (29). After dialysis into 25 mM Hepes, pH 7.5/0.5 MNaCl/1 mM DTT/1 mM EDTA/10% (wt/vol) glycerol, theprotein was frozen in liquid N2 and stored at -80°C.

Disintegration assays were as described (20). Reaction mix-tures (16 ,ul) contained 25 mM Mops (pH 7.2), 10 mM2-mercaptoethanol, 10% (wt/vol) glycerol, 100 ,g of bovineserum albumin (BSA) per ml, 7.5 mM MnCl2, 25 nM DNAsubstrate and 0.02-0.64 ,uM IN. The purified mutant IN-(50-212/F185K) was compared with the unmutagenized coredomain IN-(50-212) for disintegration activity. To allow directcomparison between IN-(50-212) and IN-(50-212/F185K),the zwitterionic detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), present in IN-(50-212) storage buffer (30), was included in disintegration assaysat a final concentration of 0.5 mM. Reactions at 37°C werestopped after 60 min with 16 ,ul of sequencing-gel loadingbuffer (95% formamide/10 mM EDTA/0.03% bromophenolblue/0.03% xylene cyanol) and 3-Al aliquots were electropho-resed in 15% polyacrylamide/urea gels.

Gel Filtration. Estimation of the molecular mass of purifiedIN-(50-212/F185K) was based on the elution time from acalibrated Superdex-75 PC 3.2/30 column (Pharmacia) on aPharmacia Smart system. The column was equilibrated prior touse with 25 mM Hepes, pH 7.5/0.5M NaCl/1mM DTT/1 mMEDTA in the presence or absence of 10 mM CHAPS. All runswere performed at 4°C with a flow rate of 30 ,ul/min.

Analytical Centrifugation. Sedimentation equilibrium ex-periments were conducted at 4°C in a Beckman Optima XL-Aanalytical ultracentrifuge, with HT-IN-(50-212/F185K) andIN-(50-212/F185K) dialyzed into 20 mM sodium phosphate,pH 7.0/0.5 M NaCl/1 mM EDTA/1 mM DTT. Data were

acquired as an average of 20 absorbance measurements at 250and 293 nm, with a radial spacing of 0.001 cm. The timerequired for the attainment of equilibrium was established byrunning at a given rotor speed until successive scans wereinvariant. Equilibrium was usually established within 18-24 hr.The data were analyzed to obtain values of the buoyantmolecular weight, M(1 - vip), using the Origin-single softwarepackage (Optima XL-A data analysis software, version 2.0;Beckman) running under Origin version 2.8. The values ofM(1- ivp) obtained from runs at 15,000, 20,000, and 25,000 rpmwere averaged to yield experimental values. Residuals werecalculated and, in all cases, a random distribution of theresiduals around zero (± <0.02) was obtained as a function ofthe radius. Values ofMwere calculated by using the density (p)of 1.020 g/ml for 0.5 M NaCl at 4.0°C obtained from standardtables. Partial specific volumes (v) of 0.737 and 0.740 ml/g forHT-IN-(50-212/F185K) and IN-(50-212/F185K), respec-tively, were calculated on the basis of amino acid composition(30).

RESULTSStrategy. We have previously examined the solubility prop-

erties of deletion derivatives of HIV-1 IN, using differentbuffer conditions, with the aim of finding fragments suitablefor physical studies. Under certain conditions, the catalyticcore domain, containing amino acids 50-212, is more solublethan full-length IN (29). However, this core domain, althoughretaining activity in the disintegration assay, exhibited aggre-gation even under conditions that improved solubility. Thisproperty impeded attempts at crystallographic studies.We postulated that the poor solubility of the core domain

might be attributable to solvent-exposed hydrophobic residues.Therefore, in an attempt to circumvent aggregation, 29 mu-tants of the core domain were made in which one or morehydrophobic residues were changed. When a single hydropho-bic amino acid was targeted, it was changed to lysine tointroduce a positive charge. When two or three hydrophobicamino acids were changed simultaneously, then the moreconservative mutation to alanine was made. Scanning-mutagenesis strategies have previously been used to investigatethe functional domains of proteins, as well as protein-proteinand protein-DNA interactions (31). These strategies haveincluded charged-to-alanine scanning and general alaninescanning. However, we are not aware of examples wheresystematic replacement of hydrophobic residues has beenemployed to improve the solubility of a protein.Mutations of IN-(50-212) That Increase Solubility. All 29

mutant proteins were expressed in E. coli and the cells werelysed with lyzozyme in either 0.15 M, 0.5 M, or 1 M NaCl. Thelysates were centrifuged, and the resultant supernatants wereanalyzed by SDS/PAGE. Substitutions of either lysine oralanine residues for hydrophobic residues within the catalyticcore domain yielded three mutant proteins with increasedsolubility (Table 1). One protein, F185K, displayed a dramaticincrease in solubility when compared with the unmutagenizedcore domain (Fig. 1). In addition, two other mutant proteins,W131A/W132A and V165K, were slightly more soluble thanthe unmutagenized core domain (data not shown).

Purification of IN-(50-212/F185K). The unmutagenizedcore domain is not soluble upon cell lysis, and purificationrequires extraction from the cell pellet with 6 M guanidinehydrochloride with subsequent refolding steps (19, 29). How-ever, the expression of HT-IN-(50-212/F185K) as a solubleprotein enabled us to successfully purify the protein undernative conditions (data not shown). HT-IN-(50-212/F185K)partitioned almost exclusively to the soluble fraction of the celllysate, and about 20 mg of protein was obtained per liter ofinduced culture. The HT was removed by cleavage withthrombin, which was subsequently removed by adsorption to

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50-212 50-212/F185Kc

X 2 a a 2 L..Iin to co~~~~~~~~~~~~ar qT. SE 0

Table 1. Solubility of point mutants of HIV-1 IN-(50-212)

Mutation(s) Solubility Mutation(s) Solubility

- 31 kDa

-21.5 kDa

- 14.4 kDa

FIG. 1. SDS/PAGE analysis of supernatants from cells expressingunmutagenized HT-IN-(50-212) or the mutant HT-IN-(50-212/F185K). Whole cell extracts of E. coli expressing HT-IN-(50-212) orHT-IN-(50-212/F185K) show induced proteins of the predicted sizethat migrate just above the 21.5-kDa molecular size marker (seearrow). Supernatants from cells lysed in 0.15, 0.5, or 1 M NaCl andsubsequently ultracentrifuged are shown in adjacent lanes.

benzamidine-Sepharose. Any residual uncleaved protein wasremoved by adsorption to a second Ni-affinity column. Afterthrombin cleavage, IN-(50-212/F185K) was soluble at >20mg/ml in 0.5 M NaCl in the absence of detergent.

Disintegration Activity. The unmutagenized core domain iscapable of catalyzing the disintegration reaction (19), althoughat reduced levels compared with the full-length protein (29).

V54A/I60AL63AL68KV75A/V77AV79KY83A/I84AV88A/189AY99A/FlOOAL1O1A/L102AL104KW108A/VllOAV113KV126KW131A/W132AW132K

+

1135KF139A/1141AY143KV15OA/I151AL158K1161A/I162AV165KL172KV176KV180A/F181A/1182AF185K1191KY194A/I200A/V201AI203A/I204A/1208A

++

Solubility of HT-IN-(50-212) mutant proteins was determined aftercell lysis. Supernatants after ultracentrifugation were analyzed bySDS/PAGE, and the intensity of the appropriate protein band wascompared with that of unmutagenized HT-IN-(50-212). -, No de-tectable increase in solubility; ±, slight increase in solubility; + + +,marked increase in solubility. The sequence of the gene encodingHT-IN-(50-212/F185K) was confirmed by DNA sequencing.

Therefore, to determine the effect of the introduced aminoacid substitution on catalytic activity, we assayed HT-IN-(50-212/F185K) and IN-(50-212/F185K) for disintegration activ-ity in parallel with the unmutagenized core domain. Both the

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HT-IN-(50-212/F185K)

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FIG. 2. Disintegration activities of unmutagenized and F185K mutant core domains. Disintegration results in the conversion of a 15-mersubstrate oligonucleotide to a 30-mer product, as shown by electrophoresis in a denaturing polyacrylamide gel. (A) Analysis of disintegrationreactions with proteins at 0.64 ,uM (based on the monomer molecular mass) with and without the HT. (B) Titration of disintegration activities.Lanes 1 and 8, 0.02 ,uM protein; lanes 2 and 9, 0.04 ALM; lanes 3 and 10, 0.08 ,AM; lanes 4 and 11, 0.12 1±M; lanes 5 and 12, 0.16 JLM; lanes 6 and13, 0.32 ,uM; lanes 7 and 14, 0.64 ,uM.

A

30-mer -_w-

15-mer -_-

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mutant and unmutagenized core domains showed similaractivities at protein concentrations of 0.64 ,uM (Fig. 2A),where the assay is saturated. However, at lower protein con-centrations, HT-IN-(50-212/F185K) (Fig. 2B) and IN-(50-212/F185K) (data not shown) were more active than the un-mutagenized core domain in the disintegration assay.IN-(50-212/F185K) Is a Monodisperse Species in Solution.

Both IN-(50-212/F185K) (Fig. 3 A and B) and HT-IN-(50-212/F185K) (data not shown) migrated as a single speciesduring gel filtration on a Superdex-75 column. In contrast,under similar conditions, unmutated IN-(50-212) was elutedas a heterogeneous series of peaks, corresponding to dimers,higher-order multimers, and large aggregates that emerged inthe void volume.The elution time of IN-(50-212/F185K), although close to

that expected for a dimer, was slightly shifted toward themonomer position (Fig. 3A). When 10 mM CHAPS wasincluded in the buffer, the elution time corresponded with thatexpected for a dimer (Fig. 3B). The retarded elution of IN-(50-212/F185K) in the absence of CHAPS is most likely dueto nonspecific interaction of the protein with the columnmatrix. This nonspecific interaction appears to be abolished byCHAPS. Unmutagenized IN-(50-212) exists as a mixture ofdimer and aggregated protein even in the presence of 10 mMCHAPS (Fig. 3C).

Analytical Centrifugation. To unambiguously determine themultimeric state, sedimentation equilibrium experiments were

0.4

0.2

0.5

81 0.25

0.2

0.1

20 30 40

Elution time, min

50

FIG. 3. Gel filtration of IN-(50-212) and IN-(50-212/F185K) ona calibrated Superdex-75 PC 3.2/30 column. (A) IN-(50-212/F185K)at 0.5 mg/ml without CHAPS in the running buffer. (B) IN-(50-212/F185K) at 0.5 mg/mlwith 10 mM CHAPS in the running buffer. (C)IN-(50-212) at 0.3 mg/ml with 10mM CHAPS in the running buffer.

performed on HT-IN-(50-212/F185K) and IN-(50-212/F185K) over a range of protein concentrations (0.6-6.0 mg/ml). An average value of 10,150 ± 280 for M(1 - vp) wasobtained for HT-IN-(50-212/F185K), corresponding to ameasured molecular weight of 40,900 ± 1000 (data not shown).The calculated molecular weight of HT-IN-(50-212/F185K) is20,040; thus, under these conditions, the protein was a dimer.For IN-(50-212/F185K) an average value of 8810 ± 70 g/molfor M(1 - vp) was obtained, corresponding to a measuredmolecular weight of 35,900 ± 300 (data not shown). Thecalculated molecular weight of IN-(50-212/F185K) is 18,159;thus, under these conditions, the protein without the HT wasalso dimeric. Molecular weight determinations at differingrotor speeds and protein concentrations revealed that, eitherwith or without a HT, IN-(50-212/F185K) appeared to be amonodisperse dimer under these buffer conditions.

DISCUSSIONOur structural studies of HIV-1 IN have focused on the centralcore domain (residues 50-212) that contains the catalytic site.Previous efforts to crystallize this core domain have beenhindered by its poor solubility and tendency to aggregate.However, a single amino acid substitution of lysine for phe-nylalanine at position 185 has produced a protein that issoluble and exists as a monodisperse dimer in solution. Thismutation has enabled the catalytic core domain of integrase tobe crystallized and the structure has been solved to 2.5-Aresolution (24).

Previous attempts at obtaining material suitable for struc-tural analysis have included extensive examination of solventconditions, generation of deletion mutants (19, 29, 32), con-struction of fusions with soluble proteins, and investigation ofIN proteins from other retroviruses. However, none of theseapproaches led to material that was suitable for crystallization.The strategy of replacing hydrophobic residues in an effort

to improve the solubility of the core domain followed thefailure of these more conventional approaches. We expectedthat many mutations we introduced would disrupt properfolding of the protein, since hydrophobic residues are ingeneral buried in proteins. However, we speculated that thefortuitous replacement of solvent-accessible hydrophobic res-idues might lead to a protein that was more soluble and lessaggregated. Improved cloning methodologies incorporatingPCR (26) made the construction and expression of a large setof mutant proteins a reasonable approach to solve this intran-sigent problem.Out of 29 mutants that were expressed, one was dramatically

more soluble and two others slightly more soluble than theunmutagenized protein. All three mutations that improved thesolubility of the core domain appear to be solvent-accessible asjudged by the crystal structure. When Trp13' and Trp132 weresimultaneously changed to alanine, there was a small increasein the solubility. When Trp132 alone was changed to lysine, noincrease in solubility was observed. These results can beexplained in terms of the crystal structure, where Trp131appears to be solvent-accessible and Trp132 is buried. Val165 isalso solvent-accessible and a change to lysine in this positionalso slightly improved the solubility of the protein. However,it is evident from the crystal structure that there are also anumber of hydrophobic residues that are solvent-accessiblewhose substitution did not lead to improved solubility.

Substitution of lysine for Phe185 resulted in a protein that wasdramatically more soluble and well behaved in solution. In thecrystal structure of IN-(50-212/F185K) there is a large dimerinterface (24). Residue 185 lies at the periphery of thisinterface and Phe185 would probably be accessible to solvent inthe unmutated protein (Fig. 4A). The absence of this solvent-exposed phenylalanine in IN-(50-212/F185K) may be suffi-cient to account for its improved solubility. However, dimer

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FIG. 4. Location of Lys185 and Ala105 in the crystal dimer ofIN-(50-212/F185K). (A) Space-filling representation of the dimerbased on the crystal structure. The monomer on the left is shown ingreen, and the monomer on the right in white. Lys'85 from the whitemonomer is blue and Ala105 from the green monomer is red. Thenitrogen of the E-amino group of Lys185 and the main-chain carbonyloxygen of Ala105 are hydrogen-bonded. In the dimer there are two suchinteractions, only one of which is visible in this representation. (B) Adetailed view of the environment around Lys185 in the crystal dimer.Red crosses mark crystallographically identified water positions. Asshown, 185 NZ, the nitrogen of the side-chain s-amino group of Lys'85on one monomer, and 105 0, the main-chain carbonyl oxygen ofAla105on the other monomer, directly interact through a hydrogen bond; 185NZ also participates in a water-mediated hydrogen bond with 133 0,

the main-chain carbonyl oxygen of Ala133 on the other monomer.Dashed lines show hydrogen bonds; distances are given in angstroms(1 A = 0.1 nm). CA, a-carbon.

stabilization may also contribute to the improved solubility ofIN-(50-212/F185K), if exclusion of the dimer interface fromsolvent favors solvation. Equilibrium centrifugation and gelfiltration studies show that IN-(50-212/F185K) is a dimer insolution, whereas the unmutated protein exists as monomers,dimers, and higher aggregates. In the crystal structure ofIN-(50-212/F185K), the side chain of Lys'85 forms a hydrogenbond across the dimer interface to the carbonyl oxygen ofAla'05 in the other subunit (Fig. 4B). Given the reasonableassumption that the dimer interface in the crystal and insolution is the same, we suggest that in IN-(50-212/F185K)

the dimer may be stabilized by this additional interaction thatis not possible with the unmutated protein.The identification of a single amino acid substitution that

results in a protein that is soluble, monodisperse, and enzy-matically active may provide a powerful tool to study thedetailed molecular mechanism of the integration reaction. Ourapproach of replacing hydrophobic residues may also be usefulin other cases where the aggregation state of a protein presentsa barrier to structure determination.

We thank D. van Gent and K. Mizuuchi for careful reading of themanuscript. We thank A. Engelman for helpful discussion and carefulreading of the manuscript. This work was supported by the NationalInstitutes of Health Intramural AIDS Targeted Antiviral Program.

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