The studies of thermal stability described above suggested that at least a portion of the gp140Δ683(−/GCN4) and gp140Δ683(−/FT) glycoproteins assumes a conformation distinct from that of a six-helix bundle. To investigate this further, we tested the ability of the five-helix protein to recognize these stabilized trimers and the gp140(−) glycoprotein. The five-helix protein consists of five of the helices (three N36 helices and two C34 helices) in the HIV-1 gp41 six-helix bundle; these five helices are joined by linker segments in the five-helix protein (35
). The five-helix protein interacts with the C34 region of the HIV-1 gp41 glycoprotein and thereby inhibits the function of the HIV-1 envelope glycoproteins but is not expected to interact with the fusion-active six-helix bundle. The His6
-tagged five-helix polypeptide was expressed in bacteria by using an expression vector kindly provided by P. Kim at the Whitehead Institute, Massachusetts Institute of Technology. About 0.5 μg of the five-helix protein in the bacterial lysates was bound to Ni-nitrilotriacetic acid (NTA) gel (Qiagen) through its His6
tag at 4°C for 16 h. After being washed with lysis buffer, the protein-gel complexes were incubated with radiolabeled gp140(−), gp140Δ683(−/GCN4), and gp140Δ683(−/FT) glycoproteins in 293T cell supernatants at 37°C for 3 h. After five washes with lysis buffer containing 15 mM imidazole, the bound glycoproteins were boiled in 1× Laemmli buffer with 2% β-mercaptoethanol (β-ME) and resolved on an SDS-7.5% polyacrylamide gel. Figure shows that all three soluble gp140 glycoproteins were recognized by the five-helix protein. Under identical conditions, a negative control protein, a His6
-tagged HIV-1 Tat protein, did not precipitate any of the soluble gp140 glycoproteins (data not shown).
FIG. 6. Recognition of soluble gp140 glycoproteins by the five-helix protein. The plasmid expressing the His6-tagged five-helix protein was used to transform Escherichia coli BL21. One of the resulting colonies was inoculated into 50 ml of Luria-Bertani medium (more ...)
The NC-1 monoclonal antibody has been shown to be relatively specific for the six-helix bundle structure of HIV-1 gp41 (27
). In our previous studies, ca. 15% of the gp140Δ683(−/GCN4) glycoprotein could bind to 1 μg of the NC-1 monoclonal antibody in a standard immunoprecipitation assay performed at room temperature for 3 h. In such an assay, the gp140Δ683(−/FT) glycoproteins bound to the NC-1 antibody at a significantly reduced level compared with the gp140Δ683(−/GCN4) trimer (Fig. , right panel). This result is consistent with the above observation with the five-helix protein, suggesting that majority of the gp140Δ683(−/FT) glycoprotein is not in a conformation containing the six-helix bundle structure, i.e., the fusogenic conformation. Moreover, the lower level of NC-1 recognition of the gp140Δ683(−/FT) glycoprotein indicates that the gp140Δ683(−/FT) trimer is more homogeneous than the gp140Δ683(−/GCN4) glycoprotein.
The availability of soluble forms of the HIV-1 envelope glycoproteins that effectively mimic the conformation of these proteins on the virus or infected cell surface is critical for attempts to obtain detailed information on the structure and function of these key molecules. The multiple oligomeric forms and instability of soluble HIV-1 gp140 preparations have created challenges for their use as reagents and as immunogens. Removal of the gp120-gp41 proteolytic cleavage site is insufficient to address these problems (6
); however, considerable increases in homogeneity result from further addition of C-terminal GCN4 trimeric motifs (48
). The trimeric globular domain of bacteriophage fibritin conferred even greater stability to heat, reducing agents, and detergents, a finding consistent with the increased stability of fibritin compared with GCN4 in other contexts (24
). The gp140Δ683(−/FT) glycoprotein exhibited greater homogeneity than the gp140Δ683(−/GCN4) trimer, as demonstrated by the reduced recognition by the NC-1 monoclonal antibody. Whether the hydrophilic surface of the fibritin trimeric motif will result in higher solubility and a lower tendency to aggregate during high-level production and concentration has yet to be tested.
The utility of soluble HIV-1 envelope glycoprotein trimers is dependent upon the extent to which the envelope glycoprotein ectodomains assume native conformations and intersubunit associations. The antigenic similarities between the gp140Δ(−/GCN4) and gp140Δ683(−/FT) glycoproteins suggest that the envelope glycoprotein ectodomains, rather than the appended trimeric motifs, dictate not only the folding of the proteins but also the orientation of the subunits within the soluble complex. Particularly reassuring was our observation that the potently neutralizing monoclonal antibodies immunoglobulin G1b12 and 2G12 exhibited a preference for binding the soluble gp140 trimers compared to the gp120 monomer. Less potently neutralizing antibodies demonstrated much less of a preference for trimer binding, and nonneutralizing antibodies bound monomeric gp120 significantly better than either of the soluble gp140 glycoprotein trimers. This finding is consistent with the expectation that the affinity of antibody binding to a structural mimic of the functional envelope glycoprotein complex should correlate with neutralization efficiency. These observed patterns of antibody recognition and the striking similarity between antigenic profiles of the gp140Δ(−/GCN4) and gp140Δ683(−/FT) trimers, in addition to the formation of intersubunit disulfide bonds in GCN4-stabilized soluble gp140 variants containing appropriately positioned cysteine substitutions (22
), suggest that at least some of the interactions among the subunits of the soluble trimers resemble those on the native HIV-1 envelope glycoproteins.
There are thought to exist different conformational states of the native HIV-1 envelope glycoproteins depending upon whether receptor has been bound and the extent of progression along the pathway leading to membrane fusion (15
). Stabilization of trimeric forms of the envelope glycoproteins could potentially increase the opportunity for the formation of the energetically favored six-helix bundle that is thought to represent a fusogenic conformation (10
). Our data suggest that at least some of the gp140Δ683(−/GCN4) and gp140Δ683(−/FT) glycoproteins exist in a conformation distinct from that of the six-helix bundle. The trimeric form of the Δ528 glycoprotein has been shown to be recognized by antibodies, like NC-1, that are specific for the six-helix bundle (29
); consistent with this, theΔ528 glycoprotein demonstrated stability at temperatures up to 85 to 95°C in our assays. At lower temperatures, the interactions among the subunits of the six-helix bundle must be energetically more favorable than the potential interactions between the monomers and the medium components, which include SDS. In contrast, significant fractions of the gp140Δ683(−/GCN4) and gp140Δ683(−/FT) glycoproteins were monomers after heating to 50 to 55°C under these conditions. The ability of the five-helix protein to precipitate the gp140Δ683(−/GCN4) and gp140Δ683(−/FT) glycoproteins suggests that the N36-binding surface of the C34 helix is accessible on these proteins, further indicating that conformations other than a six-helix bundle are assumed by these molecules. The inflections and biphasic nature of the melting curves observed for the stabilized trimers hint that these glycoproteins may undergo transitions to alternative conformations or that heterogeneous forms of these proteins exist. We expect that some of the stabilized trimers resemble the uncleaved form of the gp160 envelope glycoprotein as it exists in the Golgi apparatus prior to proteolytic activation. Further studies need to be conducted to clarify the nature of the conformational state of the stabilized soluble envelope glycoprotein trimers and to assess their suitability for detailed structural investigation. An understanding of the structure of these trimeric molecules should assist optimization of their potential as immunogens.