There is currently great interest in neutralizing antibodies that are targeted to conserved regions on E2, such as the 412–423 epitope. This region encompasses the highly conserved E2 residue W420, which plays a critical role in CD81 recognition and serves as an important contact residue for several broadly neutralizing antibodies, including AP33 and HCV1 (reviewed by Angus and Patel and Di Lorenzo et al. [3
]). Antibodies to this region are relatively uncommon during natural HCV infection, indicating that it is poorly immunogenic (51
The current study reveals the structural details of AP33 Fab in complex with its epitope peptide (Glp-LINTNGSWHVN) and allows comparison with the recently described structure of a different antibody (HCV1) in complex with a highly similar peptide (R-QLINTNGSWHIN). It is notable that the β-hairpin conformations of the peptides are very similar, despite the very different compositions of the antibody CDRs in AP33 and HCV1. This is a strong indication that the β-hairpin structure represents the conformation of this region in intact E2. Each E2 residue is buried or accessible to a similar extent whether bound to AP33 or HCV1, emphasizing the importance of residues, such as W420, which are completely buried in both complexes. It is interesting that W420 is therefore likely to be solvent exposed, despite its hydrophobic nature. However, surface-exposed tryptophans are occasionally found, particularly in proteins involved in protein-protein interactions (43
The structures of the AP33- and HCV1-bound peptide provide insights into the positioning of the epitope within the full-length glycoprotein. No high-resolution structural data currently exist for HCV E2, but Krey et al. have constructed a homology model based on related class II fusion proteins using the positions of the protein's disulfide bridges together with functional data and secondary structure predictions (27
). In the homology model, the AP33/HCV1 epitope falls within the central domain (domain I), an eight-stranded β-sandwich structure with up-and-down topology that is present in all class II fusion proteins. It is interesting to note that the epitope is predicted to encompass the majority of the first two strands, B0
, of domain I, in agreement with the AP33 and HCV1 Fab-peptide complexes, in which the peptide forms a β-hairpin, with G418 positioned at the turn. In the AP33 and HCV1 complexes, the side of the β-hairpin that lies opposite the antibody-binding face is glycosylated and therefore highly unlikely to be buried. If the peptide were a component of the β-sandwich, the antibody-binding face of the peptide, which is deeply buried when complexed with both AP33 and HCV1, would be inaccessible to antibody or CD81. In this situation, the AP33 and HCV1 antibodies would need to induce a conformational change in order to bind, incurring a high entropic cost. However, the fact that AP33 and HCV1 are both able to bind E2 with high affinity (26
; unpublished data) lends strong support to the view that the epitope exists as an exposed flap-like structure (as discussed by Kong et al. 2012 [26
])rather than forming part of the β-sandwich.
In the current study, we investigated the effects of mutations within the antibody combining site. Antibody residues that were identified from the crystal structure as being in close proximity to the epitope peptide were individually replaced with alanine. AP33 mutations that had the greatest effect on E2 binding were clustered around the central portion of the binding pocket, while more distal residues, despite being within 4.0 Å of the peptide, had little or no effect on E2 binding, suggesting that their contribution to epitope recognition is minimal. It is noteworthy that the light-chain residue YL28A did not appear to affect E2 binding, even though in the complex this tyrosine appears to interact extensively with residues toward both the N and C termini of the peptide. These results confirm that the peptide structure reflects the true conformation of this region in the intact glycoprotein and suggest that the key determinants for epitope recognition are those that anchor the central and turn regions of the β-hairpin.
The structural details of the Fab-peptide complex provide further confirmation of previous data that indicated that the linear sequence QLINTNGSWHIN (E2 residues 412 to 423) comprises the major element of the AP33 epitope. This was originally established by peptide mapping (41
), and the contact residues were identified by phage display and site-directed mutagenesis (52
). It cannot be discounted, however, that recognition by AP33 may also involve additional E2 residues beyond those represented by the linear epitope. The observation that AP33 exhibits slightly reduced binding to denatured E1E2 suggests that there may be a modest conformational component to the interaction (42
To investigate this possibility, a cross-competition analysis with well-characterized conformation-sensitive hMAbs was performed. It was surprising to note that while AP33 competed with the antigenic domain B hMAbs CBH-5, HC-1, and HC-11, there was no reduction of AP33 binding in the presence of an excess of any of the hMAbs. There are several possible interpretations of these findings. The first is that they reflect a difference in binding strengths. An antibody with high affinity will compete effectively with a low-affinity antibody but not vice versa
, with the result that nonreciprocal competition by antibodies that bind to overlapping epitopes is observed if their affinities for the antigen are markedly different. AP33 binds strongly, with an EC50
of about 0.23 nM, to genotype 1a E2 (52
; unpublished data), while the apparent affinities of HC-1, HC-11, and CBH-5 are lower, with EC50
values of 1.3 nM, 2.4 nM, and 220 nM, respectively (23
). Alternatively, if binding strengths are not too disparate, one-way competition can be an indication of close proximity, rather than overlap, of epitopes (24
). We know that the HC-11 epitope includes E2 residues 425 to 428 (25
) (), which are directly adjacent to the epitope of AP33, and that HC-1 binding is also affected by changes in this region (25
). A third possibility is that AP33, although directed primarily at the linear epitope between residues 412 and 423, has other contact residues outside this region that contribute a minor component to its epitope and are shared contact residues with antigenic domain B hMAbs. Given the conformational nature of the antigenic domain B hMAb epitopes, the availability of all discontinuous contact points may be required for binding by these antibodies, and competition for even one shared contact residue could more easily displace them. Finally, an interesting interpretation is that AP33 binding induces a conformational change in E2, as discussed above, which distorts the epitope of the antigenic domain B hMAbs sufficiently to prevent them from binding. However, this seems doubtful, since the entropic cost of such a rearrangement is likely to be prohibitively high.
To explore this further, a series of E2 alanine substitutions was tested for reactivity to AP33 and to the conformational hMAbs CBH-4D, HC-11, and CBH-7, which bind to antigenic domains A, B, and C, respectively. The results showed that E2 residues L413, N415, G418, and W420 are critical for binding, as demonstrated by a more than 73% reduction in AP33 binding of mutant compared to wild-type E2 when these residues were individually mutated to alanine. These findings correlate well with the X-ray structure, in which the corresponding four residues in the peptide are the most intimately associated with the antibody, with buried surfaces of 82%, 94%, 100%, and 99% for L413, N415, G418, and W420, respectively. Significant (>40%) reductions in binding were caused by mutations at residues 611, 614, and 652. However, these mutations also affected binding by two or three of the conformation-sensitive hMAbs, indicating that they disrupted the tertiary structure of E2. The reduction of AP33 binding by these mutations agrees with the observation that there is a conformational element to the AP33-E2 interaction. Interestingly, alanine substitution at residue L654, while not affecting antigenic domain A or domain C antibody binding, reduced binding of AP33 and HC-11 by 40%. This is the only indication of a possible contact residue shared by AP33 and the antigenic domain B hMAbs and is consistent with the observation that the adjacent residue, E655, has previously been implicated in AP33 recognition (16
). We included an E655A mutant in the analysis and saw no significant reduction in AP33 binding, in agreement with findings of the previous study, in which AP33 binding was not reduced by an E655G mutation (16
). In comparison to the effect of substituting the four critical residues L413, N415, G418, and W420, the reduction of AP33 binding by the L654A substitution is very modest, and therefore this is not strong evidence of a contact residue outside the linear epitope. That said, our analysis was limited to selected regions of E2, and there is still scope for a more extensive mutational analysis to definitively settle this question.
Residues N417 and N423 of E2 are glycosylated (17
). The exposed nature of these two asparagine side chains in the Fab-peptide complex is consistent with the observation that glycosylation at these sites does not prevent AP33 binding (52
). The structural information also sheds further light on our previous analyses of four cell culture-adaptive E2 variants that arose during extensive passaging of infected cells and exhibit enhanced in vitro
). These mutations (N415D, T416A, N417S, and I422L) occur within the AP33 epitope. The study showed that in contrast to WT E2, the N415D and N417S variants were completely resistant to neutralization by AP33 and showed greatly reduced binding to AP33 by ELISA. N415 is completely buried in the structure, and it is not unexpected that mutation of this residue in a highly shape- and charge-complementary binding site would disrupt the interaction. N417, by contrast, is exposed in the complex, and its side chain makes no contact with the antibody. However, mutation of N417 to serine is likely to introduce a new potential glycosylation site at N415 (9
). Since this residue plays such a critical role in recognition by AP33, glycosylation at N415 would certainly be expected to prevent AP33 binding. T416A and I422L remained highly sensitive to neutralization by AP33. These side chains make no direct contact with AP33 and would not be expected to greatly affect the interaction. A further mutation, G418D, was generated under AP33 selective pressure, and as with N415D and N417S, this mutant proved resistant to neutralization by AP33. G418, present at the turn of the β-hairpin, becomes buried upon AP33 binding, and the structure suggests that the interaction could not accommodate a larger side chain at this position, particularly as this would disrupt the β-hairpin.
The data presented here provide structural details of the AP33 HCV E2 epitope in its AP33-bound form and suggest that the key determinants for epitope recognition are those that anchor the central and turn regions of the β-hairpin. That the peptide displays a similar conformation whether bound to AP33 or HCV1 strongly suggests that this region does indeed form a β-hairpin in the intact glycoprotein, which is essential information for potential vaccine design. In terms of vaccine potential, the hairpin conformation opens up the possibility of designing a cyclized form of the peptide that may stabilize its secondary structure in solution.
Work toward the production of a peptide immunogen capable of eliciting AP33-like antibodies is ongoing.