Structure of the Fab Fragment of F105, a Broadly R
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病菌学杂志 2005年第20期
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717
Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe43 of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.
INTRODUCTION
A key step in the development of a successful vaccine against human immunodeficiency virus (HIV) will be the design of immunogens capable of generating an effective humoral immune response (3). Antibodies with two features characterize such a response. They must show, at the same time, both potency and broad specificity. A number of human antibodies that recognize elements of the conserved CD4 binding site of HIV type 1 (HIV-1) gp120 have now been isolated from HIV-infected patients (2, 11, 16, 24, 38, 40, 47, 49, 59, 65). This includes immunoglobulin G1 (IgG1) b12 (38, 47, 53), one of the most potent and broadly reactive anti-HIV antibodies known. In contrast, other CD4 binding-site antibodies are less potent towards many clinical isolates, even though they may be broadly cross-reactive. F105, the subject of this paper, is representative of the latter group.
F105 is an IgG1 human monoclonal antibody isolated from an HIV-infected individual (49). It binds to the CD4 binding sites of both trimeric and monomeric gp120 and is capable of neutralizing various strains of HIV (e.g., IIIB [HXBc2], MN, RF, and SF2) (7, 49, 58) but is less successful against many primary clinical isolates (12, 32). F105 did not show evidence of anti-HIV-1 activity or a viral load decrease in a phase I dose-escalation study (5, 70). However, in triple and quadruple combination therapies with anti-HIV monoclonal antibodies (2F5, 2G12, and 694/98D) with other specificities, a complete and synergistic neutralization of the SHIV-Vpu+ chimeric simian-human immunodeficiency virus was seen in macaque peripheral blood mononuclear cells in vitro and in an in vivo macaque model that mimics mucosal exposure during intrapartum virus transmission (1, 31).
Crystallographic studies of the ternary complex of the HIV gp120 core, CD4, and antibody 17b provided the first look at the structure of gp120 and its interactions with CD4 (26-28, 65). CD4 was found to bind at the nexus of the inner domain, the outer domain, and the bridging sheet of gp120 (26, 27). A large body of biochemical and biophysical data indicates a considerable conformational change in gp120 upon binding to CD4 (4, 6, 15, 24, 26, 27, 39, 41, 50, 55, 60, 62-69, 72, 73). The conformational change results in the formation and/or exposure of the chemokine receptor sites (62, 64), thus promoting further viral attachment and membrane fusion.
The molecular reorganization that results upon binding of CD4 is revealed by the structure of an unliganded simian immunodeficiency virus (SIV) gp120 core (6). With a few important exceptions, the structure of the outer domain is quite similar to that seen in the CD4-bound state. In contrast, the structure of the inner domain is markedly different. A comparison of CD4-bound and unliganded gp120 shows that the conformational change is not a simple movement of the inner domain as a rigid body. Rather, the inner domain is comprised of a set of distinct substructures that move relatively independently of one another (6). The binding of CD4 results in a rearrangement of these secondary structural elements within the inner domain (6). As predicted (24), the bridging sheet is not present in the structure of unliganded gp120 (6).
The structure of antibody b12 was determined by Saphire et al. (52-54). The structure revealed an extended CDR H3 loop with an apical tryptophan residue that is thought to recognize the Phe43 binding pocket of gp120 (53, 75). This putative interaction places significant constraints on possible gp120/b12 interactions, allowing an interaction between b12 and the CD4-bound conformation of gp120 to be modeled (53, 75). This model suggests that the broad neutralizing activity of b12 lies in its ability to interact with conserved features of the CD4 binding site through recognition of the Phe43 pocket and interactions with main chain atoms of gp120 and in its ability to recognize trimeric gp120 on the native viral surface.
A further understanding of the molecular properties that confer broad reactivity and potency upon a CD4 binding-site antibody are of substantial interest. The underlying principles are likely to impact the design of new immunogens with the potential to elicit a successful immune response. In this regard, the structure of F105 provides an opportunity to compare and contrast the structure of a broadly reactive but nonpotent CD4 binding-site antibody (F105) with that of a broadly neutralizing antibody with an overlapping epitope (b12). The comparison provides significant insight into the molecular properties that impart broad reactivity and potency upon a CD4 binding-site antibody.
MATERIALS AND METHODS
Expression and purification. The expression of F105 was performed as previously described (49). F105 was purified over protein G and eluted with 0.1 M glycine-HCl, pH 2.7, which was immediately neutralized upon collection with 1 M Tris, pH 9. Previous work had shown some contamination of protein G-purified F105 with bovine Ig from the fetal calf serum used for growing the hybridoma cells (19). Therefore, we further purified F105 using Immunopure immobilized protein L (Pierce), eluted it with 0.1 M glycine-HCl, pH 2.5, and neutralized the eluant with 1 M Na2HPO4, pH 7.2. The elution fractions containing F105 were combined and concentrated (Amicon Centricon filter with 30K molecular weight cutoff) to 4.5 mg/ml.
Fab fragments of the purified antibody were prepared by digestion with papain (0.4 μM) (ICN Biomedicals) in the presence of 20 mM ?-mercaptoethanol. The reaction was stopped by dialyzing against phosphate-buffered saline to remove the ?-mercaptoethanol, and the completeness of the digestion was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
Fab fragments were purified by anion-exchange chromatography using a POROS HQ10 column on a BioCAD Sprint perfusion chromatography system (Perseptive BioSystems, Inc.) using a linear gradient from 0 to 1.0 M NaCl in 10 mM Tris, pH 8.1. Fractions containing the F105 Fab fragment were concentrated to 6.0 mg/ml in an Amicon Ultra 10K-molecular-weight-cutoff centrifugal filter. Protein concentrations were determined by bicinchoninic acid analysis using bovine serum albumin as a standard (56). The overall yield was approximately 2.1 mg of purified Fab from 10 mg of protein G-purified antibody.
Crystallization and data collection. Purified Fab from F105 was crystallized by hanging-drop vapor diffusion at 18°C. Drops were assembled with 2 μl of F105 Fab fragment mixed with 2 μl of well solution containing 15.5% polyethylene glycol 4000, 25% isopropanol, 100 mM NaCl, and 75 mM sodium citrate, pH 5.6, with or without 1 μl of 600 μM peptide 44 (19). Crystals typically appeared in 1 to 7 weeks. Single crystals were frozen in liquid nitrogen. Data were collected at 100 K using a Rigaku RUH3R rotating anode, a CuK X-ray source, and a MAR345 image plate detector. F105 Fab fragment crystals diffracted to a 2.8-? resolution and belonged to space group P43212, with a = b = 120.4 ? and c = 72.98 ?, with a single Fab per asymmetric unit. Data were integrated and reduced using the HKL software package (42). Statistics on data completeness and quality are presented in Table 1.
Structure determination and refinement. The structure of the F105 Fab was determined by molecular replacement. Due to large variations in the elbow angles of crystallized Fabs, the molecular replacement proce
Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
We have determined the crystal structure of the Fab fragment from F105, a broadly reactive human antibody with limited potency that recognizes the CD4 binding site of gp120. The structure reveals an extended CDR H3 loop with a phenylalanine residue at the apex and shows a striking pattern of serine and tyrosine residues. Modeling the interaction between gp120 and F105 suggests that the phenylalanine may recognize the binding pocket of gp120 used by Phe43 of CD4 and that numerous tyrosine and serine residues form hydrogen bonds with the main chain atoms of gp120. A comparison of the F105 structure to that of immunoglobulin G1 b12, a much more potent and broadly neutralizing antibody with an overlapping epitope, suggests similarities that contribute to the broad recognition of human immunodeficiency virus by both antibodies. While the putative epitope for F105 shows significant overlap with that predicted for b12, it appears to differ from the b12 epitope in extending across the interface between the inner and outer domains of gp120. In contrast, the CDR loops of b12 appear to interact predominantly with the outer domain of gp120. The difference between the predicted epitopes for b12 and F105 suggests that the unique potency of b12 may arise from its ability to avoid the interface between the inner and outer domains of gp120.
INTRODUCTION
A key step in the development of a successful vaccine against human immunodeficiency virus (HIV) will be the design of immunogens capable of generating an effective humoral immune response (3). Antibodies with two features characterize such a response. They must show, at the same time, both potency and broad specificity. A number of human antibodies that recognize elements of the conserved CD4 binding site of HIV type 1 (HIV-1) gp120 have now been isolated from HIV-infected patients (2, 11, 16, 24, 38, 40, 47, 49, 59, 65). This includes immunoglobulin G1 (IgG1) b12 (38, 47, 53), one of the most potent and broadly reactive anti-HIV antibodies known. In contrast, other CD4 binding-site antibodies are less potent towards many clinical isolates, even though they may be broadly cross-reactive. F105, the subject of this paper, is representative of the latter group.
F105 is an IgG1 human monoclonal antibody isolated from an HIV-infected individual (49). It binds to the CD4 binding sites of both trimeric and monomeric gp120 and is capable of neutralizing various strains of HIV (e.g., IIIB [HXBc2], MN, RF, and SF2) (7, 49, 58) but is less successful against many primary clinical isolates (12, 32). F105 did not show evidence of anti-HIV-1 activity or a viral load decrease in a phase I dose-escalation study (5, 70). However, in triple and quadruple combination therapies with anti-HIV monoclonal antibodies (2F5, 2G12, and 694/98D) with other specificities, a complete and synergistic neutralization of the SHIV-Vpu+ chimeric simian-human immunodeficiency virus was seen in macaque peripheral blood mononuclear cells in vitro and in an in vivo macaque model that mimics mucosal exposure during intrapartum virus transmission (1, 31).
Crystallographic studies of the ternary complex of the HIV gp120 core, CD4, and antibody 17b provided the first look at the structure of gp120 and its interactions with CD4 (26-28, 65). CD4 was found to bind at the nexus of the inner domain, the outer domain, and the bridging sheet of gp120 (26, 27). A large body of biochemical and biophysical data indicates a considerable conformational change in gp120 upon binding to CD4 (4, 6, 15, 24, 26, 27, 39, 41, 50, 55, 60, 62-69, 72, 73). The conformational change results in the formation and/or exposure of the chemokine receptor sites (62, 64), thus promoting further viral attachment and membrane fusion.
The molecular reorganization that results upon binding of CD4 is revealed by the structure of an unliganded simian immunodeficiency virus (SIV) gp120 core (6). With a few important exceptions, the structure of the outer domain is quite similar to that seen in the CD4-bound state. In contrast, the structure of the inner domain is markedly different. A comparison of CD4-bound and unliganded gp120 shows that the conformational change is not a simple movement of the inner domain as a rigid body. Rather, the inner domain is comprised of a set of distinct substructures that move relatively independently of one another (6). The binding of CD4 results in a rearrangement of these secondary structural elements within the inner domain (6). As predicted (24), the bridging sheet is not present in the structure of unliganded gp120 (6).
The structure of antibody b12 was determined by Saphire et al. (52-54). The structure revealed an extended CDR H3 loop with an apical tryptophan residue that is thought to recognize the Phe43 binding pocket of gp120 (53, 75). This putative interaction places significant constraints on possible gp120/b12 interactions, allowing an interaction between b12 and the CD4-bound conformation of gp120 to be modeled (53, 75). This model suggests that the broad neutralizing activity of b12 lies in its ability to interact with conserved features of the CD4 binding site through recognition of the Phe43 pocket and interactions with main chain atoms of gp120 and in its ability to recognize trimeric gp120 on the native viral surface.
A further understanding of the molecular properties that confer broad reactivity and potency upon a CD4 binding-site antibody are of substantial interest. The underlying principles are likely to impact the design of new immunogens with the potential to elicit a successful immune response. In this regard, the structure of F105 provides an opportunity to compare and contrast the structure of a broadly reactive but nonpotent CD4 binding-site antibody (F105) with that of a broadly neutralizing antibody with an overlapping epitope (b12). The comparison provides significant insight into the molecular properties that impart broad reactivity and potency upon a CD4 binding-site antibody.
MATERIALS AND METHODS
Expression and purification. The expression of F105 was performed as previously described (49). F105 was purified over protein G and eluted with 0.1 M glycine-HCl, pH 2.7, which was immediately neutralized upon collection with 1 M Tris, pH 9. Previous work had shown some contamination of protein G-purified F105 with bovine Ig from the fetal calf serum used for growing the hybridoma cells (19). Therefore, we further purified F105 using Immunopure immobilized protein L (Pierce), eluted it with 0.1 M glycine-HCl, pH 2.5, and neutralized the eluant with 1 M Na2HPO4, pH 7.2. The elution fractions containing F105 were combined and concentrated (Amicon Centricon filter with 30K molecular weight cutoff) to 4.5 mg/ml.
Fab fragments of the purified antibody were prepared by digestion with papain (0.4 μM) (ICN Biomedicals) in the presence of 20 mM ?-mercaptoethanol. The reaction was stopped by dialyzing against phosphate-buffered saline to remove the ?-mercaptoethanol, and the completeness of the digestion was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
Fab fragments were purified by anion-exchange chromatography using a POROS HQ10 column on a BioCAD Sprint perfusion chromatography system (Perseptive BioSystems, Inc.) using a linear gradient from 0 to 1.0 M NaCl in 10 mM Tris, pH 8.1. Fractions containing the F105 Fab fragment were concentrated to 6.0 mg/ml in an Amicon Ultra 10K-molecular-weight-cutoff centrifugal filter. Protein concentrations were determined by bicinchoninic acid analysis using bovine serum albumin as a standard (56). The overall yield was approximately 2.1 mg of purified Fab from 10 mg of protein G-purified antibody.
Crystallization and data collection. Purified Fab from F105 was crystallized by hanging-drop vapor diffusion at 18°C. Drops were assembled with 2 μl of F105 Fab fragment mixed with 2 μl of well solution containing 15.5% polyethylene glycol 4000, 25% isopropanol, 100 mM NaCl, and 75 mM sodium citrate, pH 5.6, with or without 1 μl of 600 μM peptide 44 (19). Crystals typically appeared in 1 to 7 weeks. Single crystals were frozen in liquid nitrogen. Data were collected at 100 K using a Rigaku RUH3R rotating anode, a CuK X-ray source, and a MAR345 image plate detector. F105 Fab fragment crystals diffracted to a 2.8-? resolution and belonged to space group P43212, with a = b = 120.4 ? and c = 72.98 ?, with a single Fab per asymmetric unit. Data were integrated and reduced using the HKL software package (42). Statistics on data completeness and quality are presented in Table 1.
Structure determination and refinement. The structure of the F105 Fab was determined by molecular replacement. Due to large variations in the elbow angles of crystallized Fabs, the molecular replacement proce