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J. Biol. Chem., Vol. 282, Issue 43, 31803-31811, October 26, 2007
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112
From the
Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089, the Departments of Biochemistry and Molecular Genetics and ¶Pediatric Hematology-Oncology, University of Colorado Health Sciences Center, Aurora, Colorado 80045, the ||Jake Gittlen Cancer Research Foundation, Department of Pathology, Microbiology, and Immunology, Penn State College of Medicine, Hershey, Pennsylvania 17033
Received for publication, August 2, 2007 , and in revised form, September 4, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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High levels of neutralizing antibodies can be generated after immunization with HPV VLP (9). These neutralizing antibodies to L1 are highly type-specific (10). Sequence analyses of various human papillomaviruses show a high degree of homology between the major capsid proteins (L1s) of different papillomaviruses (see Fig. 1). Despite the similarity, type-specific antibody responses have been well documented in studies of genital HPV infections as well as animal model immunization studies.
Both linear and conformational epitopes have been identified on the surface of HPV L1 VLPs (11), and it is now well established that conformational epitopes are responsible for the activity of neutralizing antibody (12-14). The structure of the HPV16 T = 1 particle (VLP) was solved by Chen et al. (15), and from this structure it was suggested that the loops extending toward the outer surface of the capsomere contained the type-specific epitope(s). However, most of the HPV neutralization epitopes are conformation-dependent, and their surface localizations on VLPs as well as their amino acid compositions have not been identified. The identification of such epitopes has been done primarily by measuring the reactivity of the mAbs to the VLPs either after the site-directed mutagenesis of L1 or after the insertion of foreign peptides within the different hypervariable loops of the L1 protein (16, 17). For example, such studies suggested that an epitope composed of the FG and HI loops is immunodominant for HPV16 (18), whereas mAb binding studies for HPV6 and HPV11 have indicated that the BC, DE, and HI loops are important for these two types (19, 20).
mAb binding studies with VLPs suggest that the conformation of the surface loops play important roles in determining the type specificity of neutralizing mAbs (NmAbs) for different HPV types. To understand the interplay between type specificity of NmAbs and the conformation of the L1 surface loops at the molecular level, we have determined the L1 pentameric structures of four different HPV types HPV11, HPV16, HPV18, and HPV35. The preservation of the distinctive surface features of these pentamers unique to each type are shown by the efficient binding of the type-specific NmAbs raised against the VLPs of the corresponding HPV type. We also show that although HPV16 L1 crystal structure from the free pentamer has the same surface loop disposition as that in the previously determined T = 1 particle structure, the L1 of HPV16 and each of the other types have distinct features on their pentameric surface, unique to each HPV type, that rationalize the type specificity of the NmAbs at the atomic level.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Protein Purification—The protein expression and purification of all L1 deletion mutants was carried out essentially as described previously (15, 21, 22). Briefly, 0.2 mM isopropyl-
-D-thiogalactopyranoside was use to induce protein expression overnight at room temperature. After cell lysis by sonication in buffer L (50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTA, 10 mM phenylmethylsulfonyl fluoride), urea (ultrapure grade) was slowly added to the lysate to a final concentration of 3.0 M. The mixture was incubated at room temperature for 1 h with gentle shaking, and then dialyzed against three changes of buffer over 18 h. After centrifugation at 25,000 x g for 75 min, the supernatant was passed through a glutathione affinity column to bind GST-L1 fusion proteins. The column was washed with 10x bed volumes of buffer L to wash away contaminating proteins. L1 was then cleaved from the GST fusion using thrombin (Sigma; T6634) in an approximate ratio of 100 µg of GST-L1 to 1 NIH unit of enzyme. The digestion was carried out at 4 °C overnight. L1 pentamers were further purified by Superdex-200 (60/16 column) size exclusion chromatography.
Crystallization and Data Collection—Purified L1 pentamers of HPV11, 16, 18, and 35 were concentrated to 10-16 mg/ml in a buffer containing 20 mM Tris, pH 8.0, 50 mM NaCl, and 1 mM DTT. Crystallization trials were performed using the crystallization screens of Nextal Biotechnologies. Diffraction quality crystals were obtained by the hanging drop vapor diffusion method incubated at 18 °C for 7-10 days. HPV16 L1 was crystallized in a precipitant containing 25% polyethylene glycol 1000, 0.1 M MES, pH 6.5, and 0.1 M potassium thiocyanate. HPV18 was crystallized in the precipitant having 0.15 M sodium acetate, pH 4.6, 15% polyethylene glycol 4000, 0.16 M ammonium sulfate, 10 mM DTT, 10% glycerol, and 2.5% 1,6-hexanediol. HPV11 was crystallized in the precipitant containing 0.08 M MOPS, pH 6.5, 19% polyethylene glycol 4000, 0.4 M ammonium sulfate, 10 mM DTT, 10% glycerol, and 5% 1,6-hexanediol. HPV35 was crystallized in a precipitant containing 0.08 M sodium citrate, pH 5.6, 6% polyethylene glycol 6000, 0.45 M ammonium sulfate, 10 mM DTT, 10% glycerol. All of the crystals were flash frozen with a prior soaking in cryo-protectant containing the precipitant and 20% (v/v) glycerol.
Diffraction data for HPV11, 16, 18, and 35 L1 mutants were collected up to the resolutions of 3.2, 3.7, 3.4, and 3.5Å at synchrotron beamlines of the Advanced Light Source (Berkeley). The data were processed using HKL2000. The data statistics are given in Table 1.
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Selected mAb Binding to L1 Pentamers—Various mAbs detecting type-specific conformational and cross-reactive linear epitopes of HPV11, 16, and 18 were tested by enzyme-linked immunosorbent assay for reactivity to pentamers. The mAbs used in this study were derived from mice immunized with HPV16 and HPV18 L1 VLP antigens (18, 23) or HPV11 virions (10). All of the mAbs recognize L1 epitopes that are either conformational or linear, and the binding and neutralization properties of these mAbs have been described previously (23).
Both intact pentamers (attached to enzyme-linked immunosorbent assay plate wells at 1 µg/well in phosphate-buffered saline, pH 7.0) and denatured pentamers (attached at 1 µg/well in alkaline buffer, pH 9.5, containing dithiothreitol) were tested in duplicate. Each mAb supernatant was tested at dilutions of 1:50, and means (±S.D.) of optical densities calculated as previously described (23). Additional experiments were set up to compare binding of mAbs to pentamers versus baculovirus-derived L1 VLPs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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4; residues 404-436, HPV16 L1 numbering) (Fig. 1), were expressed as GST fusion proteins in E. coli. The 4 and the N terminus of L1 have been implicated in VLP formation (15), and the truncation of these motifs produced only pentamers of L1 that could not assemble in VLP (21). The assembly-deficient pentamers of these four types were crystallized into four different space groups (Table 1).
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Overall Pentamer Structure—L1 pentamer structures of all four HPV types grossly resemble the pentamer structure of T = 1 HPV16 L1. The HPV16 L1 free pentamer structure is shown in Fig. 2A as a representative of the four pentamer structures. In brief, five monomers are assembled in symmetrical manner to form the pentamer with a hollow channel at the center through a 5-fold central axis. The backbones of the polypeptide chain of the adjacent subunit interact directly, and the surface loops show elaborate intertwining between the individual monomers.
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Free Pentamer Structure versus the T = 1 Particle Pentamer for HPV16—To examine whether the assembly-deficient L1 pentamer has the same structure as the pentamer capable of assembly into T = 1 particles, we determined the structure of HPV16 L1 as free pentamers and compared it with the pentamer structure in the T = 1 particle (Fig. 2B). Superimposition of the core -sheets of the free pentamer structure on the pentamer of T = 1 particle revealed essentially identical conformation (root mean square deviation, 0.32 Å) not only for the core structure but also for the five surface loops (Fig. 2B), which were not used for the superposition, demonstrating that neither the internal deletion of helix 4 (4) of L1 nor the different crystal packing changed the -sheet core and more importantly the surface loop structure of HPV16 L1 pentamer. This result suggests that the loop structures are tightly anchored on to the surface of L1 pentamer, and their conformations are stably maintained as free pentamers in solution as well as in different crystal packing. The conformational integrity and stability of the assembly-deficient pentamers were also confirmed by their ability to bind a battery of NmAbs, raised against VLPs.
Binding of Free Pentamers with NmAbs Raised against VLPs of Different HPV Types—The discovery of no significant differences between the crystal structures of HPV16 L1 from the free pentamer and the pentamer in the T = 1 particle demonstrates that the helix 4 deletion, which results in free pentamers, does not lead to significant conformational changes. Therefore, it is reasonable to conclude that the surface loop conformational changes between different HPV L1 pentamers reflect the true structural differences on the viral surface of these HPV types.
To provide further experimental support for this conclusion, a panel of available NmAbs raised against VLPs was tested for binding the free pentamers (Table 2). The results showed that most of the NmAbs with conformational epitopes had strong type-specific binding with the free pentamer (Table 2), suggesting that the conformations of the viral surface loops on pentamers are maintained as in the VLPs. The linear epitope mAbs H6.C6 did not bind the pentamers, H6.C6 is known to be specific to the first 10 amino acids at the N terminus. Thus, it is expected that this mAb should not bind the free pentamer of HPV11 that lacks the first 10 N-terminal residues. H16.J4, a linear epitope mAb of HPV16, showed some weak cross-type reactivity with HPV35 L1 (Table 2). However, all the conformational epitope mAbs reacted only to the corresponding HPV types. The locations for the conformational epitopes for the NmAbs are all accessible from the top surface of L1 pentamers for the different HPV types.
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We have performed a pairwise structural comparison of HPV11, HPV18, and HPV35 pentamers with the pentamer taken from HPV16 T = 1 particle by superposition based on the two core -sheets from each monomer.
HPV11 versus HPV16 L1 Pentamers—The pentameric structure of HPV11 L1 was compared with that of HPV16, and the root mean square deviation of the superposition was 0.38 Å, indicating that the -sheet core structures are almost identical for HPV11 and HPV16 (Fig. 3A). However, structural differences for the loops, located on the pentamer surface, are obvious in the superposition (Fig. 3, B and C). The BC and HI loops of HPV11 are 3.5 and 2.0 Å shorter than the corresponding loops of HPV16 L1 (Fig. 3B), which correlates with the amino acid deletion in these two loops of HPV11 L1 (Fig. 1). Such structural differences of the two loops are expected to exhibit distinct protein-protein interaction specificity in their antibody binding.
Structural differences are also observed in the DE loop (Fig. 3C), and distinct amino acid sequence differences between the two types were observed. For example, the residues corresponding to Gly133, Gly134, and Pro136 on the DE loop of HPV11 L1 are all Ala in HPV16 (Fig. 1). Because Gly and Pro are known to exert local structural perturbation, because of either the known flexibility or lack of an amide hydrogen, the observed conformational difference of the DE loop of HPV11 L1 from that of HPV16 L1 is expected in this case. However, the EF loop conformation of HPV11 L1 is similar to that of HPV16 L1 (Fig. 3D).
HPV18 Versus HPV16 L1 Pentamers—The -sheet core of the HPV18 L1 pentamer superimposes well with HPV16 L1, with a root mean square deviation of 0.47 Å. However, substantial structural differences were observed for all five surface loops (Fig. 4). The BC loop of HPV18 L1 shows a drastically different conformation, with its tip oriented 
13 Å away from the corresponding loop of HPV16 L1 (Fig. 4B). The deviation of BC loop of HPV18 starts from Pro53, unique to HPV18, whereas the deviation ends at Pro63 conserved in all four types. This conformational shift of the BC loop has been accommodated through a lateral displacement of the adjacent EF loop, moving in a concerted manner in the same direction, where the residues of the EF loop have been shifted by 4 Å from that of HPV16 (Fig. 4B). This shift of BC and EF loops in HPV18 L1 places the two loops in close contact, allowing the formation of a number of hydrogen bonding interactions between the residues from the BC and EF loops (Fig. 4B).
The conformation of the HI loop of HPV18 L1 is significantly different from the HI loop of HPV16 L1 (Fig. 4C), which may be the result of the combined effect of an amino acid insertion and the presence of two Pro residues at successive positions (351 and 353) in HPV18 L1 compared with HPV16. Interestingly, the HI loop in HPV11, 16, and 18 forms hydrogen bonds with the EF on one side and with FG loops on the other side (Fig. 4C), which may help stabilize the HI loop conformation on the surface.
The DE loop is located on the inner channel ridge of the L1 pentamer. The diameter of the pentamer channel is 18 Å for HPV16 L1, but it narrows down to 10 Å in HPV18 L1 (Fig. 4D). This narrowing of the pentameric channel in HPV18 L1 is caused by the shifting of DE loop toward the channel center. The residue His133 located at the narrowest point of the channel is protruding into the channel space (Fig. 4D).
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sheet core of HPV16 L1 produced a root mean square deviation of 0.38 Å, indicating a very similar core structure (Fig. 5A). However, the superposition reveals different structural features for the surface loops. The FG loop of HPV35 has a two-residue deletion compared with HPV16 (as well as HPV11 and HPV18) (Fig. 1), which results in the structural difference of this loop (Fig. 5B). The region of the DE loop in proximity of the FG loop also shows a structural change compared with HPV16. It appears that the 133-141 region of the DE loop of HPV35 L1 moves toward the FG loop (Fig. 5B). Structural differences are observed for both the BC and EF loops between HPV 35 and HPV16 (Fig. 5C). Subtle but significant conformational changes are observed for the HI loop. In addition to the conformational difference, the orientation of different amino acid side chains on the tip of HI loop accounts for the major differences between these two HPV types (Fig. 5D). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-sheet core structures of the L1 pentamers of the four HPV types are nearly identical, the loop structures on the pentamer surfaces display significant conformational differences (Figs. 3, 4 and 5). The high type specificity of the conformational epitope NmAbs we tested here supportively suggests that a few angstrom shift of the surface loop or a few substitutions of the surface loop residues can lead to changes of the viral surface antigen determinants in eliciting different antibodies, as well as to the disruption of binding with a mAb that recognizes the same location on the pentamer surface of another HPV type.
Pairwise comparisons reveal that the extent of conformational changes for a particular loop is different for different pairs. The loops with a few insertions or deletions of amino acids expectedly showed moderate spatial shifts (of 4 Å), but the extent and the direction of large structural differences (that range up to 7-15 Å), as observed in HPV18 and HPV35, were not predictable from their primary sequences. The conformational differences of the surface loops can also be visualized in the surface representation of the pentamer structures in Fig. 6. Because most of the known epitopes for the available NmAbs are located on the surface loops, the distinct structural features of these surface loops might be expected to form distinct surface conformational epitopes.
A minor structural difference of the epitope loop, with a spatial shift of 2 Å, is found to be sufficient to disrupt the antibody-epitope binding. Such a disruption of antibody binding is further amplified by the substitution of one or more residues on the loops from different HPV types. This change is evident in the structural differences in the DE loop between HPV11 and HPV16, where a combination of the structural shift of the DE loop (Fig. 3) with a three-residue substitution from Gly-Gly-Pro in HPV11 to Ala-Ala-Ala in HPV16 produced a rather distinct structural feature for the DE loop (Figs. 3 and 6), abrogating the binding of HPV11 DE loop specific NmAbs, H11.F1, and H11.G5 to HPV16.
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Similarly, FG loop of HPV35 posses a distinguished conformation compared with the FG loop of HPV16, which contains a known epitope for HPV16.E70 (Fig. 7A). Rather, the orientation of the late region of FG loop coupled with the orientation of adjacent DE loop form a distinct structural feature. The distance between these loops widely vary in the four HPV types being the maximum (15 Å) in HPV18 and the minimum (7 Å) in HPV35. A notable conformational difference is also observed at the tip of the HI loop of the four types (Fig. 7C) that contains a known epitope for NmAb H11.H3. These phenomena may collectively explain why no cross-reactivity between HPV types is observed for the NmAbs with conformational epitopes on the viral surface.
However, there are parts on the pentamer surface area, such as the base of the HI loop and the early region of the FG loop (Fig. 7D), which have essentially identical structures among the different types. If epitopes in these areas can elicit antibody production as strong antigen determinants, these epitopes are expected to be cross-reactive between types and offer cross-protection as vaccine. Nonetheless, no cross-reactive NmAbs with conformational epitopes have been identified yet, which may suggest that these "common" epitopes may not have strong antigenicity or may be sterically inaccessible. In conclusion, despite the high sequence homology of L1 proteins between different types and the overall structural conservation of L1 pentamers, the detailed loop structures on the pentamer (and thus viral) surface are largely different because of their different amino acid composition and insertions/deletions, which not only change the surface epitope composition and conformation but also affect the viral surface antigenicity, providing a molecular rationalization for the type specificity of NmAbs.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed: Dept. of Molecular and Computational Biology, University of Southern California, 1050 Childs Way, RRH201, Los Angeles, CA 90089. Tel.: 213-740-5487; Fax: 213-740-4390; E-mail: Xiaojiang.Chen{at}USC.edu/.
3 The abbreviations used are: HPV, human papillomavirus; mAb, monoclonal antibody; VLP, virus-like particle; NmAb, neutralizing mAb; GST, glutathione S-transferase; DTT, dithiothreitol; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) at the N terminus, C terminus, and helix 4 are deleted in our constructs.
