Catalytic domain of human immunodeficiency virus type 1

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

Catalytic domain of human immunodeficiency virus type 1 integrase: Identification of a soluble mutant by systematic replacement of hydrophobic residues TIMOTHY M. JENKINS, ALISON B. HICKMAN, FRED DYDA, RODOLFO GHIRLANDO, DAVID R. DAVIES, ROBERT CRAIGIE*

AND

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

reaction (19). This catalytic domain, which is relatively resistant to proteolysis (20), contains the conserved residues AspM6, Asp116, and Glu152, the D,D-35-E motif found in all retroviral integrases. These acidic residues, which also form the same motif in all retrotransposons and certain bacterial transposable elements, may coordinate metal ions directly involved in

ABSTRACT The integrase protein of human immunodeficiency virus type 1 is necessary for the stable integration of the viral genome into host DNA. Integrase catalyzes the 3' processing of the linear viral DNA and the subsequent DNA strand transfer reaction that inserts the viral DNA ends into host DNA. Although full-length integrase is required for 3' processing and DNA strand transfer activities in vitro, the central core domain of integrase is sufficient to catalyze an apparent reversal of the DNA strand transfer reaction, termed disintegration. This catalytic core domain, as well as the full-length integrase, has been refractory to structural studies by x-ray crystallography or NMR because of its low solubility and propensity to aggregate. In an attempt to improve protein solubility, we used site-directed mutagenesis to replace hydrophobic residues within the core domain with either alanine or lysine. The single substitution of lysine for phenylalanine at position 185 resulted in a core domain that was highly soluble, monodisperse in solution, and retained catalytic activity. This amino acid change has enabled the catalytic domain of integrase to be crystallized and the structure has been solved to 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 residues may be a useful strategy to improve the solubility of other proteins to facilitate structural and biochemical studies.

catalysis (20-23). The propensity of IN to aggregate at concentrations below those required for many physical methods has frustrated structural and biophysical studies. In particular, aggregation has prevented structural analysis by x-ray crystallography or NMR. We report that a single amino acid substitution, Phe185 -- Lys (F185K), in the catalytic core domain of HIV-1 IN results in a protein that is dramatically more soluble, is monodisperse in solution, and retains catalytic activity. The mutation has enabled this domain to be crystallized and the structure has been solved to 2.5-A resolution (24). MATERIALS AND METHODS Construction of Site-Directed Mutant Plasmids. General techniques for manipulating DNA were as described (25). DNA fragments encoding site-directed mutations were generated by an "overlapping" PCR protocol (26), using Vent DNA polymerase (New England Biolabs) according to the manufacturer's guidelines. Primers were designed to generate DNA fragments containing an Nde I site at the 5' termini and a stop codon flanked by a BamHI site at the 3' termini. These PCR fragments were produced in a two-step procedure. In a first round of PCR, DNA fragments were generated by using plasmid pINSD (20) as template DNA. Amplification using the 5' (Nde I site) primer with a 3' primer containing a mutation produced a PCR "halffragment." Separate amplification with the 3' (BamHI site) primer and the 5' primer containing the mutation produced the other half-fragment. In a second round of PCR, the two overlapping half-fragments, after gel purification, were mixed together with the two external restriction site-containing primers for a final PCR to generate a full-length DNA fragment containing the desired mutation. After gel purification and cleavage with BamHI and Nde I, the full-length DNA fragments were ligated into pET-15b (Novagen), placing the site-directed mutants under the control of a T7 promoter (27). This vector encodes a 20-aa histidine tag (HT) at the amino terminus

Retroviral replication requires the integration of a linear double-stranded DNA copy of the viral RNA genome into a host cell chromosome (1, 2). Prior to integration, cleavage of the 3' ends of the linear viral DNA removes two nucleotides 3' of a conserved CA dinucleotide. The processed 3' ends of the viral DNA are then covalently joined to host DNA in a subsequent DNA strand transfer reaction. In the resulting integration intermediate (3, 4), the 5' ends of the viral DNA and the 3' ends of the host DNA at the site of integration remain unjoined. Host enzymes are probably responsible for repairing the single-strand connections between viral and host DNA to complete the integration process. Regions of the viral genome that are essential for integration map to the 3' end of the 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 strand transfer reactions in vitro with oligonucleotide substrates that mimic the ends of HIV-1 DNA (13-15). IN can also catalyze an apparent reversal of the strand transfer reaction, termed disintegration (16). Deletion studies on HIV-1 and HIV-2 IN have revealed that although both the 3' processing and DNA strand transfer reactions require a full-length protein (17, 18), the central core domain is able to catalyze the disintegration

(MGSSHHHHHHSSGLVPRGSH-) that allows rapid purification of the expressed protein on a nickel-chelating column. This sequence also contains a thrombin cleavage site (LVPRGS) to allow removal of the HT after purification. Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate; DTT, dithiothreitol; HIV-1, human immunodeficiency virus type 1; HT, histidine tag; IN, integrase. *To whom reprint requests should be addressed at: Building 5, Room 301, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; electronic mail-mail:[email protected].

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

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Protein Expression and Solubility Measurements. Plasmids encoding the site-directed mutants were expressed in Escherichia coli BL21(DE3) (28). Protein expression was induced as described (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 of Super broth (Biofluids) containing 100 ,ug of ampicillin per ml were resuspended in 12 ml of 25 mM Hepes (pH 7.5) and frozen in liquid N2. Resuspended cells (100 ,ul) were lysed by the 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 of 170 ,ul. Cells were incubated 30 min on ice, frozen in liquid N2, and thawed. The lysate was centrifuged in a Beckman TL-100 ultracentrifuge for 45 min at 100,000 x g and the supernatant

recovered. Ten microliters of supernatant was mixed with 2 ,ul of protein sample loading buffer [0.36 M Tris HCl, pH 6.8/10% (wt/vol) SDS/40% (wt/vol) glycerol/50 mM DTT/ 0.005% (wt/vol) bromophenol blue], heated at 100°C for 5 min, and analyzed by SDS/PAGE. Protein was detected by staining with Coomassie blue R-250. Protein Purification and Disintegration Activity. Cells expressing the mutant that contains the single substitution of lysine for phenylalanine at position 185 [HT-IN-(50-212/ F185K)] were harvested from a 1 liter culture and suspended in 12 ml of 25 mM Hepes (pH 7.5) before freezing in liquid N2. Thawed cells were suspended in lysis buffer (25 mM Hepes, pH 7.5/0.5 M NaCl/2 mM 2-mercaptoethanol/5 mM imidazole with lysozyme at 0.3 mg/ml) to a final volume of 40 ml. Lysed cells were sonicated and then centrifuged at 30,000 x g for 40 min. The supernatant was filtered through a 0.45-,um filter and was

applied

Proc. Natl. Acad. Sci. USA 92

Biochemistry: Jenkins et al.

to a

Ni-affinity (chelating Sepharose; Pharmacia)

column (0.5 x 8 cm). After extensive washing with 20 mM and 60 mM imidazole in elution buffer [25 mM Hepes, pH 7.5/0.5 M NaCl/2 mM 2-mercaptoethanol/10% (wt/vol) glycerol], protein was eluted with a 10-column-volume linear gradient of 60 mM to 1 M imidazole in elution buffer. Pooled fractions containing HT-IN-(50-212/F185K) were dialyzed against 25 mM Hepes, pH 7.5/0.5 M NaCl/1 mM DTT/1 mM EDTA/ 10% (wt/vol) glycerol. The HT was removed by cleavage with thrombin (29). After dialysis into 25 mM Hepes, pH 7.5/0.5 M NaCl/1 mM DTT/1 mM EDTA/10% (wt/vol) glycerol, the protein was frozen in liquid N2 and stored at -80°C. Disintegration assays were as described (20). Reaction mixtures (16 ,ul) contained 25 mM Mops (pH 7.2), 10 mM 2-mercaptoethanol, 10% (wt/vol) glycerol, 100 ,g of bovine serum albumin (BSA) per ml, 7.5 mM MnCl2, 25 nM DNA substrate and 0.02-0.64 ,uM IN. The purified mutant IN-(50212/F185K) was compared with the unmutagenized core domain IN-(50-212) for disintegration activity. To allow direct comparison between IN-(50-212) and IN-(50-212/F185K), the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), present in IN-(50212) storage buffer (30), was included in disintegration assays at a final concentration of 0.5 mM. Reactions at 37°C were stopped after 60 min with 16 ,ul of sequencing-gel loading buffer (95% formamide/10 mM EDTA/0.03% bromophenol blue/0.03% xylene cyanol) and 3-Al aliquots were electrophoresed in 15% polyacrylamide/urea gels. Gel Filtration. Estimation of the molecular mass of purified IN-(50-212/F185K) was based on the elution time from a calibrated Superdex-75 PC 3.2/30 column (Pharmacia) on a Pharmacia Smart system. The column was equilibrated prior to use with 25 mM Hepes, pH 7.5/0.5 M NaCl/1 mM DTT/1 mM EDTA in the presence or absence of 10 mM CHAPS. All runs were performed at 4°C with a flow rate of 30 ,ul/min. Analytical Centrifugation. Sedimentation equilibrium experiments were conducted at 4°C in a Beckman Optima XL-A analytical ultracentrifuge, with HT-IN-(50-212/F185K) and dialyzed into 20 mM sodium phosphate, IN-(50-212/F185K) pH 7.0/0.5 M NaCl/1 mM EDTA/1 mM DTT. Data were

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acquired as an average of 20 absorbance measurements at 250 and 293 nm, with a radial spacing of 0.001 cm. The time required for the attainment of equilibrium was established by running at a given rotor speed until successive scans were invariant. Equilibrium was usually established within 18-24 hr. The data were analyzed to obtain values of the buoyant molecular weight, M(1 - vip), using the Origin-single software package (Optima XL-A data analysis software, version 2.0; Beckman) running under Origin version 2.8. The values of M(1 - ivp) obtained from runs at 15,000, 20,000, and 25,000 rpm were averaged to yield experimental values. Residuals were calculated and, in all cases, a random distribution of the residuals around zero (± 20 mg/ml in 0.5 M NaCl in the absence of detergent. Disintegration Activity. The unmutagenized core domain is capable of catalyzing the disintegration reaction (19), although at reduced levels compared with the full-length protein (29).

Therefore, to determine the effect of the introduced amino acid substitution on catalytic activity, we assayed HT-IN-(50212/F185K) and IN-(50-212/F185K) for disintegration activity in parallel with the unmutagenized core domain. Both the

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FIG. 2. Disintegration activities of unmutagenized and F185K mutant core domains. Disintegration results in the conversion of a 15-mer substrate oligonucleotide to a 30-mer product, as shown by electrophoresis in a denaturing polyacrylamide gel. (A) Analysis of disintegration reactions 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 and 13, 0.32 ,uM; lanes 7 and 14, 0.64 ,uM.

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mutant and unmutagenized core domains showed similar activities at protein concentrations of 0.64 ,uM (Fig. 2A), where the assay is saturated. However, at lower protein concentrations, HT-IN-(50-212/F185K) (Fig. 2B) and IN-(50-212/ F185K) (data not shown) were more active than the unmutagenized 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-(50212/F185K) (data not shown) migrated as a single species during gel filtration on a Superdex-75 column. In contrast, under similar conditions, unmutated IN-(50-212) was eluted as a heterogeneous series of peaks, corresponding to dimers, higher-order multimers, and large aggregates that emerged in

the void volume. The elution time of IN-(50-212/F185K), although close to that expected for a dimer, was slightly shifted toward the monomer position (Fig. 3A). When 10 mM CHAPS was included in the buffer, the elution time corresponded with that expected for a dimer (Fig. 3B). The retarded elution of IN(50-212/F185K) in the absence of CHAPS is most likely due to nonspecific interaction of the protein with the column matrix. This nonspecific interaction appears to be abolished by CHAPS. Unmutagenized IN-(50-212) exists as a mixture of dimer and aggregated protein even in the presence of 10 mM CHAPS (Fig. 3C). Analytical Centrifugation. To unambiguously determine the multimeric state, sedimentation equilibrium experiments were 0.4

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FIG. 3.

Gel filtration of

50

40

30

time,

IN-(50-212)

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and

IN-(50-212/F185K)

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Superdex-75 PC 3.2/30 column. (A) IN-(50-212/F185K) mg/ml without CHAPS in the running buffer. (B) IN-(50-212/

a calibrated

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F185K)

at 0.5

IN-(50-212)

mg/mlwith 10 mM CHAPS in the running buffer. (C) mg/ml with 10 mM CHAPS in the running buffer.

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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) was obtained for HT-IN-(50-212/F185K), corresponding to a measured molecular weight of 40,900 ± 1000 (data not shown). The calculated molecular weight of HT-IN-(50-212/F185K) is 20,040; thus, under these conditions, the protein was a dimer. For IN-(50-212/F185K) an average value of 8810 ± 70 g/mol for M(1 - vp) was obtained, corresponding to a measured molecular weight of 35,900 ± 300 (data not shown). The calculated molecular weight of IN-(50-212/F185K) is 18,159; thus, under these conditions, the protein without the HT was also dimeric. Molecular weight determinations at differing rotor speeds and protein concentrations revealed that, either with or without a HT, IN-(50-212/F185K) appeared to be a monodisperse dimer under these buffer conditions.

DISCUSSION Our structural studies of HI V-1 IN have focused on the central core domain (residues 50-212) that contains the catalytic site. Previous efforts to crystallize this core domain have been hindered by its poor solubility and tendency to aggregate. However, a single amino acid substitution of lysine for phenylalanine at position 185 has produced a protein that is soluble and exists as a monodisperse dimer in solution. This mutation has enabled the catalytic core domain of integrase to be crystallized and the structure has been solved to 2.5-A resolution (24). Previous attempts at obtaining material suitable for structural analysis have included extensive examination of solvent conditions, generation of deletion mutants (19, 29, 32), construction of fusions with soluble proteins, and investigation of IN proteins from other retroviruses. However, none of these approaches 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 the failure of these more conventional approaches. We expected that many mutations we introduced would disrupt proper folding of the protein, since hydrophobic residues are in general buried in proteins. However, we speculated that the fortuitous replacement of solvent-accessible hydrophobic residues might lead to a protein that was more soluble and less aggregated. Improved cloning methodologies incorporating PCR (26) made the construction and expression of a large set of 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 the unmutagenized protein. All three mutations that improved the solubility of the core domain appear to be solvent-accessible as judged by the crystal structure. When Trp13' and Trp132 were simultaneously changed to alanine, there was a small increase in the solubility. When Trp132 alone was changed to lysine, no increase in solubility was observed. These results can be explained in terms of the crystal structure, where Trp131 appears to be solvent-accessible and Trp132 is buried. Val165 is also solvent-accessible and a change to lysine in this position also slightly improved the solubility of the protein. However, it is evident from the crystal structure that there are also a number of hydrophobic residues that are solvent-accessible whose substitution did not lead to improved solubility. Substitution of lysine for Phe185 resulted in a protein that was dramatically more soluble and well behaved in solution. In the crystal structure of IN-(50-212/F185K) there is a large dimer interface (24). Residue 185 lies at the periphery of this interface and Phe185 would probably be accessible to solvent in the unmutated protein (Fig. 4A). The absence of this solventexposed phenylalanine in IN-(50-212/F185K) may be sufficient to account for its improved solubility. However, dimer

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Proc. Natl. Acad. Sci. USA 92 (1995)

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the dimer may be stabilized by this additional interaction that is not possible with the unmutated protein. The identification of a single amino acid substitution that results in a protein that is soluble, monodisperse, and enzymatically active may provide a powerful tool to study the detailed molecular mechanism of the integration reaction. Our approach of replacing hydrophobic residues may also be useful in other cases where the aggregation state of a protein presents a barrier to structure determination. We thank D. van Gent and K. Mizuuchi for careful reading of the manuscript. We thank A. Engelman for helpful discussion and careful reading of the manuscript. This work was supported by the National Institutes of Health Intramural AIDS Targeted Antiviral Program. 1. Whitcomb, J. M. & Hughes, S. H. (1992) Annu. Rev. Cell Bio. 8, 275-306. 2. Goff, S. P. (1992) Annu. Rev. Genet. 26, 527-544. 3. Fujiwara, T. & Mizuuchi, K. (1988) Cell 54, 497-504. 4. Brown, P. O., Bowerman, B., Varmus, H. E. & Bishop, J. M. (1989) Proc. Natl. Acad. Sci. USA 86, 2525-2529. 5. Donehower, L. A. & Varmus, H. E. (1984) Proc. Natl. Acad. Sci. USA 81, 6461-6465. 6. Hippenmeyer, P. J. & Grandgenett, D. P. (1984) Virology 137, 358-362. 7. Panganiban, A. T. & Temin, H. M. (1984) Proc. Natl. Acad. Sci. USA 81, 7885-7889. 8. Schwartzberg, P., Colicelli, J. & Goff, S. P. (1984) Cell 37, 1043-1052. 9. Panganiban, A. T. & Temin, H. M. (1983) Nature (London) 306, 155-160. 10. Colicelli, J. & Goff, S. P. (1985) Cell 42, 573-580. 11. Cobrinik, D., Katz, R., Terry, R., Skalka, A. M. & Leis, J. (1987) J. Virol. 61, 1999-2008. 12. Colicelli, J. & Goff, S. P. (1988) J. Mol. Biol. 199, 47-59. 13. Sherman, P. A. & Fyfe, J. A. (1990) Proc. Natl. Acad. Sci. USA

FIG. 4. Location of Lys185 and Ala105 in the crystal dimer of IN-(50-212/F185K). (A) Space-filling representation of the dimer based on the crystal structure. The monomer on the left is shown in green, and the monomer on the right in white. Lys'85 from the white monomer is blue and Ala105 from the green monomer is red. The nitrogen of the E-amino group of Lys185 and the main-chain carbonyl oxygen of Ala105 are hydrogen-bonded. In the dimer there are two such interactions, only one of which is visible in this representation. (B) A detailed view of the environment around Lys185 in the crystal dimer. Red crosses mark crystallographically identified water positions. As shown, 185 NZ, the nitrogen of the side-chain s-amino group of Lys'85 on one monomer, and 105 0, the main-chain carbonyl oxygen of Ala105 on the other monomer, directly interact through a hydrogen bond; 185 NZ 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 of IN-(50-212/F185K), if exclusion of the dimer interface from solvent favors solvation. Equilibrium centrifugation and gel filtration studies show that IN-(50-212/F185K) is a dimer in solution, whereas the unmutated protein exists as monomers, dimers, and higher aggregates. In the crystal structure of IN-(50-212/F185K), the side chain of Lys'85 forms a hydrogen bond across the dimer interface to the carbonyl oxygen of Ala'05 in the other subunit (Fig. 4B). Given the reasonable assumption that the dimer interface in the crystal and in solution is the same, we suggest that in IN-(50-212/F185K)

87, 5119-5123. 14. Bushman, F. D. & Craigie, R. (1991) Proc. Natl. Acad. Sci. USA 88, 1339-1343. 15. Lafemina, R. L., Callahan, P. L. & Corgingley, M. G. (1991) J. Virol. 65, 5624-5630. 16. Chow, S. A., Vincent, K. A., Ellison, V. & Brown, P. 0. (1992) Science 255, 723-726. 17. Drelich, M., Wilhelm, R. & Mous, J. (1992) Virology 137, 358-362. 18. Vink, C., Oude Groeneger, A. A. M. & Plasterk, R. H. A. (1993) Nucleic Acids Res. 21, 1419-1425. 19. Bushman, F. D., Engelman, A., Palmer, I., Wingfield, P. & Craigie, R. (1993) Proc. Natl. Acad. Sci. USA 90, 3428-3432. 20. Engelman, A. & Craigie, R. (1992) J. Virol. 66, 6361-6369. 21. Van Gent, D. C., Oude Groeneger, A. M. & Plasterk, R. H. (1992) Proc. Natl. Acad. Sci. USA 89, 9598-9602. 22. Kulkosky, J., Jones, K. S., Katz, R. A., Mack, J. P. G. & Skalka, A. M. (1992) Mol. Cell. Biol. 12, 2331-2338. 23. Khan, E., Mack, J. P., Katz, R. A., Kulkosky, J. & Skalka, A. M.

(1991) Nucleic Acids Res. 19, 851-860. 24. Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266, 1981-1986. 25. Maniatis, T., Fritsch, E. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press,

Plainview, NY). 26. Higuchi, R., Krummel, B. & Saiki, R. K. (1988)NucleicAcidsRes. 16, 7351-7367. 27. Studier, F. W. & Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130. 28. Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S. M., Dunn, J. J. & Studier, F. W. (1987) Gene 56, 125-135. 29. Hickman, A. B., Palmer, I., Engelman, A., Craigie, R. & Wingfield, P. (1994) J. Biol. Chem. 269, 29279-29287. 30. Perkins, S. J. (1986) Eur. J. Biochem. 15, 169-180. 31. Wells, J. A. (1991) Methods Enzymol. 202, 390-411. 32. Engelman, A., Bushman, F. D. & Craigie, R. (1993) EMBO J. 12, 3269-3275.