Mapping the Epitope of an Inhibitory Monoclonal Antibody to the C-terminal DNA-binding Domain of HIV-1 Integrase
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m105072200
ISSN1083-351X
AutoresJizu Yi, Hong Cheng, Mark Andrake, Roland L. Dunbrack, Heinrich Röder, Anna Marie Skalka,
Tópico(s)HIV Research and Treatment
ResumoIntegrase (IN) catalyzes the insertion of retroviral DNA into chromosomal DNA of a host cell and is one of three virus-encoded enzymes that are required for replication. A library of monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1) IN was raised and characterized in our laboratory. Among them, monoclonal antibody (mAb) 33 and mAb32 compete for binding to the C-terminal domain of the HIV-1 IN protein. Here, we show that mAb33 is a strong inhibitor of IN catalytic activity, whereas mAb32 is only weakly inhibitory. Furthermore, as the Fab fragment of mAb32 had no effect on IN activity, inhibition by this mAb may result solely from its bivalency. In contrast, Fab33 did inhibit IN catalytic activity, although bivalent binding by mAb33 may enhance the inhibition. Interaction with Fab33 also prevented DNA binding to the isolated C-terminal domain of IN. Results from size-exclusion chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses revealed that multiple Fab33·IN C-terminal domain complexes exist in solution. Studies using heteronuclear NMR showed a steep decrease in1H-15N cross-peak intensity for 8 residues in the isolated C-terminal domain upon binding of Fab33, indicating that these residues become immobilized in the complex. Among them, Ala239 and Ile251 are buried in the interior of the domain, whereas the remaining residues (Phe223, Arg224, Tyr226, Lys244, Ile267, and Ile268) form a contiguous, solvent-accessible patch on the surface of the protein likely including the epitope of Fab33. Molecular modeling of Fab33 followed by computer-assisted docking with the IN C-terminal domain suggested a structure for the antibody-antigen complex that is consistent with our experimental data and suggested a potential target for anti-AIDS drug design. Integrase (IN) catalyzes the insertion of retroviral DNA into chromosomal DNA of a host cell and is one of three virus-encoded enzymes that are required for replication. A library of monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1) IN was raised and characterized in our laboratory. Among them, monoclonal antibody (mAb) 33 and mAb32 compete for binding to the C-terminal domain of the HIV-1 IN protein. Here, we show that mAb33 is a strong inhibitor of IN catalytic activity, whereas mAb32 is only weakly inhibitory. Furthermore, as the Fab fragment of mAb32 had no effect on IN activity, inhibition by this mAb may result solely from its bivalency. In contrast, Fab33 did inhibit IN catalytic activity, although bivalent binding by mAb33 may enhance the inhibition. Interaction with Fab33 also prevented DNA binding to the isolated C-terminal domain of IN. Results from size-exclusion chromatography, gel electrophoresis, and matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses revealed that multiple Fab33·IN C-terminal domain complexes exist in solution. Studies using heteronuclear NMR showed a steep decrease in1H-15N cross-peak intensity for 8 residues in the isolated C-terminal domain upon binding of Fab33, indicating that these residues become immobilized in the complex. Among them, Ala239 and Ile251 are buried in the interior of the domain, whereas the remaining residues (Phe223, Arg224, Tyr226, Lys244, Ile267, and Ile268) form a contiguous, solvent-accessible patch on the surface of the protein likely including the epitope of Fab33. Molecular modeling of Fab33 followed by computer-assisted docking with the IN C-terminal domain suggested a structure for the antibody-antigen complex that is consistent with our experimental data and suggested a potential target for anti-AIDS drug design. Integrase (IN) 1The abbreviations used are: INintegraseHIV-1human immunodeficiency virus type 1mAbmonoclonal antibodySPRsurface plasmon resonanceELISAenzyme-linked immunosorbent assayHSQCheteronuclear single-quantum correlationMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight is one of three virus-encoded enzymes that are required for retroviral replication (1.Katz R.A. Skalka A.M. Annu. Rev. Biochem. 1994; 63: 133-173Crossref PubMed Scopus (535) Google Scholar). IN catalyzes insertion of the linear double-stranded viral DNA into the chromosomal DNA of a host cell. The IN protein comprises three distinct regions known as the N-terminal, catalytic core, and C-terminal domains (see Fig. 1A) (2.Andrake M.D. Skalka A.M. J. Biol. Chem. 1996; 271: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 3.Hindmarsh P. Leis J. Microbiol. Mol. Biol. Rev. 1999; 63: 836-843Crossref PubMed Google Scholar). The isolated N-terminal domain (residues 1 to ∼50) assumes a three-helix bundle structure with a helix-turn-helix motif stabilized by Zn2+ coordination that stimulates enzymatic activity in vitro (4.Cai M. Zheng R. Caffrey M. Craigie R. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1997; 4: 567-577Crossref PubMed Scopus (306) Google Scholar, 5.Eijkelenboom A.P. van den Ent F.M. Vos A. Doreleijers J.F. Hard K. Tullius T.D. Plasterk R.H. Kaptein R. Boelens R. Curr. Biol. 1997; 7: 739-746Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The N-terminal domain contributes to formation of tetrameric or higher multimeric forms of the protein (6.Zheng R. Jenkins T.M. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13659-13664Crossref PubMed Scopus (309) Google Scholar, 7.Lee S.P. Han M.K. Biochemistry. 1996; 35: 3837-3844Crossref PubMed Scopus (68) Google Scholar, 8.Lee S.P. Xiao J. Knutson J.R. Lewis M.S. Han M.K. Biochemistry. 1997; 36: 173-180Crossref PubMed Scopus (157) Google Scholar). Crystallographic analysis of the catalytic core domains of human immunodeficiency virus type 1 (HIV-1) IN and avian sarcoma virus IN revealed that each subunit binds at least one divalent cation cofactor, Mg2+ or Mn2+, mediated by acidic residues in the highly conserved D,D(35)E motif that comprises the active site of the enzyme (9.Goldgur Y. Dyda F. Hickman A.B. Jenkins T.M. Craigie R. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9150-9154Crossref PubMed Scopus (371) Google Scholar, 10.Maignan S. Guilloteau J.P. Zhou-Liu Q. Clement-Mella C. Mikol V. J. Mol. Biol. 1998; 282: 359-368Crossref PubMed Scopus (265) Google Scholar, 11.Bujacz G. Jaskolski M. Alexandratos J. Wlodawer A. Merkel G. Katz R.A. Skalka A.M. Structure. 1996; 4: 89-96Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 12.Bujacz G. Alexandratos J. Wlodawer A. Merkel G. Andrake M. Katz R.A. Skalka A.M. J. Biol. Chem. 1997; 272: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Binding of the metal cofactor to this motif activates HIV-1 IN (13.Asante-Appiah E. Skalka A.M. J. Biol. Chem. 1997; 272: 16196-16205Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 14.Asante-Appiah E. Seeholzer S.H. Skalka A.M. J. Biol. Chem. 1998; 273: 35078-35087Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and stimulates preferential attachment of the protein to its viral DNA substrate (15.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar). The C-terminal domain, comprising amino acids 220–270, binds DNA. Its NMR structure resembles that of the SH3 domain, a motif known to promote protein-protein interactions (16.Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (271) Google Scholar, 17.Eijkelenboom A.P. Puras-Lutzke R.A. Boelens R. Plasterk R.H. Kaptein R. Hard K. Nat. Struct. Biol. 1995; 2: 807-810Crossref PubMed Scopus (219) Google Scholar, 18.Eijkelenboom A.P. Sprangers R. Hard K. Puras-Lutzke R.A. Plasterk R.H. Boelens R. Kaptein R. Proteins Struct. Funct. Genet. 1999; 36: 556-564Crossref PubMed Scopus (80) Google Scholar). Although the isolated C-terminal domain forms a homodimer in solution, only the catalytic core domain forms a dimer in the crystal structure of a two-domain derivative of HIV-1 IN that includes the core and C-terminal domains (19.Chen J.C.-H. Krucinski J. Miercke L.J. Finer-Moore J.S. Tang A.H. Leavitt A.D. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8233-8238Crossref PubMed Scopus (383) Google Scholar). In such crystals, the C-terminal domain moieties are separated from each other by 55 Å, but are involved in a variety of contacts with C-terminal domains in adjacent unit cells. integrase human immunodeficiency virus type 1 monoclonal antibody surface plasmon resonance enzyme-linked immunosorbent assay heteronuclear single-quantum correlation matrix-assisted laser desorption ionization time-of-flight IN proteins must recognize and bind to both viral and host cell target DNAs. In the processing reaction, two nucleotides are removed from the 3′-ends of the viral DNA. These new 3′-ends are then joined to phosphorus atoms in the backbone of both strands of the target DNA in a concerted cleavage and ligation reaction. The strongest DNA-binding determinants of HIV-1 IN have been localized to the C-terminal domain, but such binding is sequence-independent. Despite intense efforts in many laboratories, the detailed mechanism by which IN interacts with host and viral DNAs remains unknown. To address this and other questions of structure and function, a library of monoclonal antibodies (mAbs) was raised against HIV-1 IN and characterized in our laboratory (20.Bizub-Bender D. Kulkosky J. Skalka A.M. AIDS Res. Hum. Retroviruses. 1994; 10: 1105-1115Crossref PubMed Scopus (49) Google Scholar). Several of these mAbs inhibit the enzymatic activities of IN in vitro. Among these, mAb17 binds to the N-terminal domain, mAb4 binds to the catalytic core, and mAb32 and mAb33 bind to the C-terminal domain of IN (see Fig. 1A). Details of the binding of the N-terminal domain to the inhibitory mAb17 have been reported (21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); its epitope was mapped to a relatively neutral surface of the dimeric N-terminal domain, likely to be involved in protein-protein interaction. Here, we describe detailed binding studies and structural analyses of the interaction of the C-terminal domain-specific mAb33. We show that binding of Fab33 prevented the interactions of the C-terminal domain with DNA substrates and also inhibited the enzymatic activity of full-length IN, whereas binding of Fab32 produced neither effect. The epitope recognized by Fab33 was mapped to the three-dimensional structure of IN-(220–270) using heteronuclear NMR spectroscopy and computer-assisted molecular modeling. Construction of plasmid pET29b, encoding wild-type HIV-1 IN, IN-F185K/C280S, and IN-3CS (with three Cys → Ser substitutions at positions 56, 65, and 280), was reported previously (13.Asante-Appiah E. Skalka A.M. J. Biol. Chem. 1997; 272: 16196-16205Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 14.Asante-Appiah E. Seeholzer S.H. Skalka A.M. J. Biol. Chem. 1998; 273: 35078-35087Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar). For NMR experiments, the protein expression vectors pET28b/IN-(220–270) and pET28b/IN-(220–288) were prepared by inserting sequences encoding amino acids 220–270 and 220–288 of HIV-1 IN, respectively, into plasmid pET28b (Novagen, Madison, WI) as described (21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The products include four expression plasmid-encoded amino acids (GSHM) at their N termini, which remain after thrombin cleavage to remove the His6 tag. 15N-Labeled IN-(220–270) and 2H,15N-labeled IN-(220–288) were purified from Escherichia coli strain BL21(DE3) grown in M9 minimal medium with [15N]ammonium chloride as the sole nitrogen source and in H2O or 99% D2O. As higher yield and better purification were obtained with IN-(220–288), this protein was used for NMR analysis of the mAb33 epitope. Procedures for protein expression and purification, monoclonal antibody preparation, and isolation of Fab fragments were reported previously (21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22.Yi J. Skalka A.M. Biopolymers. 2000; 55: 308-318Crossref PubMed Scopus (11) Google Scholar). The effect of mAb32 and mAb33 on the enzymatic activity of full-length HIV-1 IN was determined by measuring both the processing and joining reactions using a 21-base pair oligonucleotide duplex that represents the viral U5 DNA end as substrate (13.Asante-Appiah E. Skalka A.M. J. Biol. Chem. 1997; 272: 16196-16205Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Assay conditions are described in the relevant figure legends. Surface plasmon resonance (SPR; BIACORE) was employed to analyze the interactions between the isolated C-terminal domain of IN and DNA substrates. Double-stranded oligonucleotides representing either the viral U5 DNA end (20-mer) or a target DNA substrate (24-mer) with no sequence match with viral DNA ends were immobilized on the surface of a chip as described (15.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar). To allow association, solutions of the IN protein were applied to a chip containing immobilized DNA. Dissociation of IN from the nucleoprotein complex was monitored in real-time after application of buffer to wash the chip. The kinetic rate constants for dissociation (koff or kd) and for apparent association (kon or ka) were obtained by fitting the real-time data, and the apparent dissociation constant was calculated as Kd =koff/kon (15.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar). The HIV-1 IN-3CS protein was immobilized on 96-well high binding microtiter plates by applying a solution containing 1 μg IN/well in a total volume of 50 μl of Tris-buffered saline (20 mm Tris-HCl (pH 7.5) and 150 mm NaCl). After overnight incubation, 50 μl of 1 mg/ml bovine serum albumin in the above buffer was added to each well, and the plates were incubated for 2 h to block the remaining binding sites. The plates were subsequently washed with 200 μl of Tris-buffered saline four times, followed by addition of primary antibodies and secondary antibodies labeled with horseradish peroxidase. The standard protocol was then followed, and the relative binding efficiency of monoclonal antibody to the immobilized IN protein was determined by measurement of absorbance at 405 nm (13.Asante-Appiah E. Skalka A.M. J. Biol. Chem. 1997; 272: 16196-16205Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 20.Bizub-Bender D. Kulkosky J. Skalka A.M. AIDS Res. Hum. Retroviruses. 1994; 10: 1105-1115Crossref PubMed Scopus (49) Google Scholar, 21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). NMR spectra were recorded at 37 °C on a Bruker DMX 600-MHz spectrometer equipped with a 5-mm x, y, z-shielded pulsed-field gradient triple-resonance probe. Typically, each sample contained ∼0.5 mm IN-(220–288) dissolved in 95% H2O and 5% D2O at a final buffer concentration of 50 mmNaH2PO4 (pH 6.5), 100 mm NaCl, and 0.5 mm EDTA, which was the same as that used by Lodi et al. (16.Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (271) Google Scholar). Protein-mAb interaction was studied by recording 1H,15N HSQC NMR spectra (23.Kay L.E. Keifer P. Saarine T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2439) Google Scholar) for each sample of IN-(220–288) mixed with Fab33 at Fab/IN ratios of 0.16:1.0, 0.35:1.0, 0.55:1.0, 0.75:1.0, and 0.98:1.0. To minimize line broadening due to dipole-dipole coupling, the IN protein was uniformly labeled with 2H in addition to 15N (24.Huang X. Yang X. Luft B.J. Koide S. J. Mol. Biol. 1998; 281: 61-67Crossref PubMed Scopus (35) Google Scholar). Each1H,15N HSQC spectrum was recorded as a 2048 (1H) × 128 (15N) data matrix with acquisition times of 285 ms in t2 and 78 ms in t1. The data were acquired with 32 scans for each hypercomplex t1/t2increment with an interscan delay of 1 s. The total acquisition time was 3.5 h for each spectrum. The NMR data were processed using XWINNMR2 (Bruker) and analyzed using Felix2000 NMR processing software (MSI). The 1H,15N HSQC spectrum of IN-(220–270) was assigned on the basis of the work of Lodi et al. (16.Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (271) Google Scholar) and Clore. 2G. M. Clore, personal communication. Thirty-nine well resolved peaks in the HSQC spectrum of IN-(220–288) were assigned by comparison. (The remaining resonances were obscured by the signals from the C-terminal tail (residues 271–288).) As a qualitative measure of the line broadening due to addition of Fab33, we determined peak heights for resolved cross-peaks in the base line-corrected HSQC spectra. Peak heights show a more linear dependence on the concentration ratio of Fab33 to IN-(220–288) than peak volumes. Solvent-accessible surface areas were calculated using GETAREA Version 1.1 (Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX) (25.Fraczkiewicz R. Braun W. J. Comp. Chem. 1998; 19: 319-333Crossref Scopus (883) Google Scholar). MALDI-TOF mass spectrometric analysis was carried out as described previously (21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22.Yi J. Skalka A.M. Biopolymers. 2000; 55: 308-318Crossref PubMed Scopus (11) Google Scholar). Slight modifications in experimental conditions are described in the relevant figure legends. The assay was performed on a Superdex 200/PC3.2 column (3.2 mm × 30 cm) equilibrated with 50 mm Hepes (pH 7.5) containing 300 mm NaCl. The column was calibrated with standard proteins of known molecular masses (Bio-Rad), thyroglobulin (670 kDa), γ-globulin (158 kDa), bovine serum albumin (68 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa), using linear regression on a plot of log molecular mass versus elution time. Samples to be analyzed (each protein at 120 μm) were incubated for 30 min on ice prior to injection and eluted at 0.2 ml/min. The eluted proteins were monitored by measuring the absorbance at 280 or 220 nm for IN-(220–288). IN-(220–288) was mixed with Fab33 and incubated in the presence of 2.5 mm glutaraldehyde cross-linker in 20 mm Hepes (pH 7.5) and 500 mmNaCl for 30 min at 25 °C. Each protein was present at a final concentration of 28 μm in the 1:1 ratio reaction. Reactions were terminated by quenching with 40 mm glycine for 10 min prior to addition of loading buffer for analysis by SDS-PAGE. Equal samples of each reaction were loaded on a 16% Tris/glycine gel prior to visualization by silver staining. Modeling of the Fv fragment of mAb33 was performed as described previously (21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) using PSI-BLAST to compare the sequences of the heavy and light chain variable domains with sequences of known structures in the Protein Data Bank (codes 1IBG and 1A7O, respectively). Sequence identity was 49% for the heavy chain and 62% for the light chain. Side chain coordinates were predicted with the SCWRL program (26.Bower M.J. Cohen F.E. Dunbrack Jr., R.L. J. Mol. Biol. 1997; 267: 1268-1282Crossref PubMed Scopus (488) Google Scholar). As described previously (21.Yi J. Arthur J.W. Dunbrack Jr., R.L. Skalka A.M. J. Biol. Chem. 2000; 275: 38739-38748Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) the program HEX Version 2.3 (27.Ritchie D.W. Kemp G.J.L. Proteins Struct. Funct. Genet. 2000; 39: 178-194Crossref PubMed Scopus (492) Google Scholar) 3Available at www.biochem.abdn.ac.uk. was used to identify a reasonable structure for the complex. The calculations were started in a conformation with the antibody combining site and residues of IN-(220–270) (Protein Data Bank code 1QMC, Ref. 18.Eijkelenboom A.P. Sprangers R. Hard K. Puras-Lutzke R.A. Plasterk R.H. Boelens R. Kaptein R. Proteins Struct. Funct. Genet. 1999; 36: 556-564Crossref PubMed Scopus (80) Google Scholar) identified as potential points of contact, pointed toward one another. The proteins were then allowed to move in a 60° arc (±30°) in each direction and ±8 Å along the intermolecular axis to produce the best fit. The solvent-accessible surface area buried upon Fab·IN complex formation is calculated as the difference between the accessible surface area of complexed IN-(220–270) and free IN-(220–270). Our previous studies showed that the activity of HIV-1 IN is stimulated by preincubation with the divalent metal ion (Mg2+ or Mn2+) required for catalysis (13.Asante-Appiah E. Skalka A.M. J. Biol. Chem. 1997; 272: 16196-16205Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Such stimulation is correlated with a conformational change that is blocked by the binding of mAb33 to the apoenzyme. mAb33 also inhibits the processing, joining, and disintegration activities of HIV-1 IN (14.Asante-Appiah E. Seeholzer S.H. Skalka A.M. J. Biol. Chem. 1998; 273: 35078-35087Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Our previous analyses also showed that mAb33 and mAb32 compete for binding to the C-terminal region of the HIV-1 IN protein (20.Bizub-Bender D. Kulkosky J. Skalka A.M. AIDS Res. Hum. Retroviruses. 1994; 10: 1105-1115Crossref PubMed Scopus (49) Google Scholar). Furthermore, the binding of Fab32 has been mapped to specific residues in the C-terminal domain based on MALDI-TOF mass spectrometric analysis combined with time-limited proteolysis (22.Yi J. Skalka A.M. Biopolymers. 2000; 55: 308-318Crossref PubMed Scopus (11) Google Scholar). To further investigate the binding of mAb33, we first performed immunoblot analyses with full-length HIV-1 IN (residues 1–288) and four different truncated proteins: IN-(50–212) (the isolated catalytic core domain), IN-(50–288) (the catalytic core and C-terminal domains), IN-(220–270) (the C-terminal domain only), and IN-(220–288) (the C-terminal domain and the “tail”) (Fig. 1B). The results show that mAb33 bound only to fragments containing the C-terminal domain (amino acids 220–270) and that neither the C-terminal region of the core domain (IN-(50–212)) nor the tail of the C-terminal domain (IN-(271–288)) contributed significantly to recognition of mAb33, confirming and extending our previous observations (20.Bizub-Bender D. Kulkosky J. Skalka A.M. AIDS Res. Hum. Retroviruses. 1994; 10: 1105-1115Crossref PubMed Scopus (49) Google Scholar). Thus, for further analysis of the mAb33-binding site, we used IN-(220–270) and the more soluble IN-(220–288) interchangeably. To determine whether the effects on IN enzymatic activity are related to the ability of the bivalent antibodies to cross-link the protein, we compared the binding affinities and inhibitory activities of the intact antibodies (mAb32 and mAb33) with the corresponding Fab fragments (Fab32 and Fab33). ELISA studies revealed that mAb33 and Fab33 formed stable complexes with full-length HIV-1 IN-3CS, with half-saturation concentrations of 3.0 × 10−9 and 3.0 × 10−8m, respectively (Fig. 1C). In contrast, there was no significant difference in the binding of mAb32 and Fab32 to HIV-1 IN-3CS; half-saturation concentrations were 5.0 × 10−7 and 4.0 × 10−7m, respectively (Fig. 1D). However, these values are ∼10-fold higher than the dissociation constant determined for Fab33 and ∼100-fold higher than that of mAb33. Kinetic analysis using SPR showed that Fab33 formed a stable complex with IN-(220–270). The off-rate constant was 4.0 × 10−3 s−1; the on-rate constant was 5.0 × 105 s−1m−1; and the dissociation constant (Kd) was ∼8 nm. These data are consistent with results from ELISAs shown in Fig. 1C. The effects of the mAbs and Fab fragments on HIV-1 IN processing activity were tested by quantitation of the −2 cleavage (processing) product from a 21-base pair oligodeoxynucleotide duplex that represents the viral U5 DNA end. The results show that both mAb33 and Fab33 inhibited HIV-1 IN processing (Fig. 2A) and joining (data not shown) activities. The half-inhibition concentrations (IC50) of mAb33 and Fab33 were 0.18 and 1.8 μm, respectively. The observation that mAb33 exhibited ∼10-fold higher inhibitory activity than its Fab fragment is consistent with the difference observed in the binding affinities of mAb33 and Fab33 (Fig. 1C). We conclude that bivalent binding is not required for inhibition by mAb33, but may enhance the inhibitory effect. These results are consistent with our previous observations that intracellular expression of the single-chain variable region of this antibody (single-chain Fv33) in a target cell prevents HIV-1 infection (28.Levy-Mintz P. Duan L. Zhang H. Hu B. Dornadula G. Zhu M. Kulkosky J. Bizub-Bender D. Skalka A.M. Pomerantz R.J. J. Virol. 1996; 70: 8821-8832Crossref PubMed Google Scholar). Thus, the in vivo activity of single-chain Fv33 is correlated with the ability of Fab33 to inhibit IN enzymatic activity in vitro. On the other hand, mAb32 had a weak inhibitory effect (IC50 ∼ 2.0 μm), and Fab32 had no detectable effect on the processing activity of IN (Fig. 2A), although the binding affinities of these two reagents for IN were approximately equal (Fig. 1C). Thus, the inhibitory activity probably reflects the ability of the bivalent mAb32 to hold two IN molecules together in a nonproductive complex. Binding of the isolated catalytic core domain of HIV-1 IN to duplex oligodeoxynucleotide DNA substrates was not detectable in our previous studies using SPR (15.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar). In contrast, the quantitative analysis shown in Fig. 2B indicated a dissociation constant (Kd) of 1.5 μm for a duplex oligodeoxynucleotide representing the viral U5 DNA end in complex with the isolated C-terminal fragment IN-(220–288). The same Kd value was observed with IN-(220–270) (data not shown). These results indicated that as with mAb33 binding (Fig. 1B), the C-terminal 18-amino acid tail (amino acids 271–288) made no significant contribution to the stability of the DNA·IN-(220–288) complex. Furthermore, when a nonviral DNA substrate was immobilized, the Kd value measured was 1.7 μm, confirming that DNA sequence does not affect the affinity of the C-terminal domain interactions with DNA (29.Woerner A.M. Klutch M. Levin J.G. Marcus-Sekura C.J. AIDS Res. Hum. Retroviruses. 1992; 8: 297-304Crossref PubMed Scopus (73) Google Scholar, 30.Vink C. Oude Groeneger A.M. Plasterk R.H. Nucleic Acids Res. 1993; 21: 1419-1425Crossref PubMed Scopus (247) Google Scholar, 31.Puras-Lutzke R.A. Vink C. Plasterk R.H. Nucleic Acids Res. 1994; 22: 4125-4131Crossref PubMed Scopus (173) Google Scholar). As both mAb33 and mAb32 bind to the C-terminal domain of HIV-1 IN, we investigated whether these mAbs could affect the ability of IN to bind to DNA substrates. Results of SPR measurements indicated that binding of the C-terminal domain to an immobilized DNA substrate was inhibited by preincubation of the protein with Fab33 (Fig. 2C); the binding profile for Fab33 + IN-(220–288) was indistinguishable from that of Fab33 alone. Detailed studies at various Fab33 concentrations indicated that this inhibition increased with increasing Fab33/IN ratio (data not shown). Complete inhibition was achieved at a Fab/IN-(220–288) ratio of 1:1 (as illustrated in Fig. 2C) or higher (data not shown). However, if IN-(220–288) was preincubated with equimolar amounts of Fab32, we observed an increase in the signal (Table I). No such increase was observed upon preincubation of IN-(220–288) with a nonspecific mAb (mouse anti-MOPC21). The relative response/resonance unit increase observed in the presence of Fab32 can be explained by the higher molecular mass of the Fab32·IN-(220–288) complex compared with IN-(220–288) alone. These results suggest that the Fab32·IN-(220–288) complex can bind to the immobilized DNA substrate, whereas binding of Fab33 to the C-terminal domain of IN inhibits the protein-DNA interaction.Table IEffects of mAb33 and mAb32 on binding of IN-(220–288) to DNA substratesAntibodyRelative RUDNA binding%Model viral DNA None100+ Anti-MOPC2198 ± 3+ Fab330.2 ± 1.0− Fab32194 ± 10+Model target DNA None100+ Anti-MOPC21101 ± 5.0+ Fab330.0 ± 1.0− Fab32121 ± 8+Data were obtained from experiments similar to those described for Fig. 2C. After incubation in either the absence or presence of equimolar concentrations of mAb or Fab, IN-(220–288) was injected on the chip surface containing immobilized viral U5 model DNA substrate or model target DNA substrate. The response units (RU) were read after protein injection (15.Yi J. Asante-Appiah E. Skalka A.M. Biochemistry. 1999; 38: 8458-8468Crossref PubMed Scopus (51) Google Scholar). Open table in a new tab Data were obtained from experiments similar to those described for Fig. 2C. After incubation in either the absence or presence of equimolar concentrati
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