Characterization of a Conformational Epitope on Hepatitis B Virus Core Antigen and Quasiequivalent Variations in Antibody Binding

We have characterized a conformational epitope on capsids ofhepatitis B virus (HBV) by cryo-electron microscopy and three-dimensionalimage reconstruction of Fab-labeled capsids to $~$ 10-Å resolution,combined with molecular modeling. The epitope straddles theinterface between two adjacent subunits and is discontinuous,consisting of five peptides—two on one subunit and threeon its neighbor. Together, the two icosahedral forms of theHBV capsid—T=3 and T=4 particles—present seven quasiequivalentvariants of the epitope. Of these, only three bind this Fab.Occupancy ranges from $~$ 100 to $~$ 0%, reflecting conformational variationsin the epitope and steric blocking effects. In the former, smallshifts of the component peptides have large effects on bindingaffinity. This approach appears to hold general promise forelucidating conformational epitopes of HBV and other viruses,including those of neutralizing and diagnostic significance.

INTRODUCTION

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Introduction

Antibody-combining sites on proteins are of two types, calledlinear and conformational epitopes, respectively (37). Linearepitopes typically consist of 6 to 12 consecutive amino acidresidues: antibodies that recognize them also bind to the peptidesin question and to the denatured protein, for example, in Westernblots. However, some linear peptides are not recognized on thenative protein because they occupy sites that are inaccessibleto antibodies (cryptic epitopes). Conformational epitopes, onthe other hand, are presented only when the antigen assumesits native conformation and are not recognized on peptides ordenatured proteins. Most such epitopes, also called discontinuousepitopes (1), consist of two or more peptides from differentparts of the polypeptide chain spatially juxtaposed by the protein'sthree-dimensional fold.

Complex antigens such as viral capsids potentially contain manyepitopes. In the course of a natural infection, however, thenumber of immunodominant epitopes tends to be limited. The factorsinvolved in selecting them are incompletely understood. Themajority of epitopes on native (i.e., folded) protein antigensare thought to be conformational (4). These include many clinicallyimportant viral epitopes, such as the capsid-associated coreantigen of hepatitis B virus (HBcAg) (30) and surface antigen(sAg), its envelope glycoprotein (12). An important goal forvaccine development is to be able to characterize immunodominantconformational epitopes, both in terms of the contributing peptidesand structurally, in order to ultimately engineer their presentationon alternative platforms as synthetic antigens.

Linear epitopes may be mapped on the antigen's amino acid sequenceby testing the reactivity of defined fragments with the antibodyin question. This operation may be done systematically withthe Pepscan technique, using a set of overlapping peptides thatspans the entire sequence (18). Conformational epitopes cannotbe characterized in this way, and mapping inferences tend tobe indirect, based on binding competitions with other antibodiesthat have known linear epitopes. However, this approach is subjectto pitfalls: antibodies are large molecules, and binding ofan antibody to one site can block access of a second antibodyto other sites considerably removed in space and greatly displacedalong the antigen's primary sequence or even located on anothersubunit.

Conformational epitopes may be identified from crystal structuresof the antigen complexed with Fab fragments of a monoclonalantibody, and such identifications have been achieved in a fewcases (e.g., see references 24 and 32). However, this approachimposes daunting requirements in terms of the amount of material,crystallinity, and data analysis. Here we demonstrate an alternativeapproach based on cryo-electron microscopy (cryo-EM) of Fab-decoratedantigens (31, 38), which requires less material by >3 ordersof magnitude and has no need for crystals. The basic idea isas follows: provided that the antigen structure is known tohigh resolution, a cryo-EM structure of the antigen-Fab complexat moderate resolution, probed by molecular modeling with ageneric Fab structure from the protein database, contains sufficientinformation to allow identification of the peptides that makeup the epitope. We demonstrate proof of principle by using thisapproach to dissect the binding of monoclonal antibody 3120,which is specific for a hitherto unidentified conformationalepitope on hepatitis B virus (HBV) capsids (35).

MATERIALS AND METHODS

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Materials and Methods

Preparation of HBcAg capsids. Capsids were prepared essentially as described (39). ConstructCp149.3CA consists of residues 1 to 149 in which the cysteinesat positions 48, 61, and 107 have been changed to alanines.Capsids recovered from bacterial extracts by gel filtrationwere dissociated with 1.5 M urea at pH 9.5 into dimeric proteinand further purified by gel filtration. Reassembly was inducedby either dilution or dialysis into 100 mM HEPES-350 mM NaCl,pH 7.0. Capsids were freed of unassembled protein and bufferexchanged by gel filtration with 50 mM HEPES, 100 mM NaCl, pH8.0. The particles were concentrated by ultrafiltration to 2mg/ml. Protein concentration was determined by absorbance at280 nm corrected for light scattering ( ${varepsilon}$ ₂₈₀ = 29,500 M^-1 ·cm^-1).

Generation of Fabs and decoration of capsids. Monoclonal antibody (MAb 3120) was purchased from the Instituteof Immunology, Tokyo, Japan. To produce Fab fragments, MAbsat 0.5 mg/ml were first reduced by adding EDTA to 1 mM and TCEP(Tris[2-carboxyethylphosphine] hydrochloride) (Pierce, Rockford,Ill.) to 3.5 mM and incubating for 0.5 h at room temperature.Immobilized papain resin (Pierce) was equilibrated with 10 mMEDTA-3.5 mM TCEP · HCl-20 mM PO₄, pH 7.0. Antibody (55µl) and centrifugally drained resin (15 µl) weremixed and incubated for 1 h at 37°C with tumbling. The resinwas removed by centrifugal filtration. Fabs were mixed withcapsids at a 2:1 ratio of Fab:dimer and incubated overnightat room temperature. Fc fragments were removed by adding 0.1volume of settled immobilized protein G resin (Pierce), incubatingfor 0.5 h at room temperature, pelleting the resin in a microcentrifuge,and recovering the supernatant. The volume of the sample wasreduced under a stream of nitrogen gas until a particle densitysatisfactory for cryo-electron microcopy was attained.

Cryo-EM. Data were collected according to Cheng et al. (13). In brief,specimens were vitrified in thin films suspended over holeycarbon films, and micrographs were recorded at a magnificationof x38,000 on a CM200-FEG microscope (FEI, Eindhoven, Netherlands)fitted with a Gatan 626 cryoholder, operating at 120 keV. Focalpairs of micrographs were recorded at $~$ 10 e^-/Å² per exposure.The first exposure was defocused such that the first zero ofthe contrast transfer function was at a frequency of (17 Å)^-1- (20 Å)^-1. For the second exposure, the defocus was increasedby 0.4 µm.

Image reconstruction. Six focal pairs were digitized with a SCAI scanner (Z/I Imaging,Huntsville, Ala.), giving 7-µm pixels (1.8 Å atthe specimen). Density maps were calculated with the PFT algorithm(2) as described previously (16), using preexisting maps ofCp147 capsids (T=4[15] and T=3[13]) as starting models. Thereconstructions included all particles with correlation coefficientsabove a threshold of (mean - 1 standard deviation) for eachof the three parameters calculated by PFT. Specifically, thethreshold values were as shown in Table 1. Thus, the T=4 mapused 1,710 out of 2,758 originally picked particles and hada resolution of 10.1 Å (Fourier shell correlation threshold,0.3). The T=3 map used 479 particles out of 839 and had a resolutionof 13.8 Å.

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TABLE 1. Threshold values for each of three correlation coefficients calculated by PFT

Molecular modeling. Coordinates of HBV capsid protein (PDB 1QGT [43]) were fittedinto cryo-EM envelopes of T=3 and T=4 reconstructions by visualcriteria (39), using the program O (21). The coordinates ofan immunoglobulin G2a (IgG2a) Fab-peptide antigen complex (PDB1FPT [40]) were then fitted into the Fab portion of the T=4HBV-Fab 3120 density map at the site overlying the C-D interface(see Fig. 1 for designation of A, B, C, D—the four quasiequivalentsubunits per T=4 capsid). Colores (11) was then used to refinethe fits of the HBV subunits into both capsid reconstructionsand of Fab 1FPT into the capsid-Fab 3120 reconstruction. TheFab coordinates were split into antigen-binding and non-antigen-bindingdomains to allow variation in the elbow angle. To save computationtime and avoid redundant solutions, only the portion of thereconstruction immediately surrounding each subunit being fitted(2 to 3 Å beyond the surface of the volume determinedby visual fitting) was included in the Colores fitting. Theresulting T=4 coordinates differed by root-mean-square deviationsof 1.0 and 3.2 Å from the "by-eye" and 1QGT coordinates,respectively. To test the robustness of our 1FPT fit, additionalIgG2a structures—1FOR (23), 1AIF (3), and 1BOG (22) werefitted into the Fab density via Colores. The root mean square(RMS) differences between the three additional fits and the1FPT fit were 0.9 to 1.5 Å as measured from the C ${alpha}$ positionsof all residues in ß-strands of the antigen-binding-domain,or 1.1 to 1.8 Å from the C ${alpha}$ positions of all residues notin variable-length loops.

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FIG. 1. Cryo-EM reconstructions of HBV capsids labeled with Fab 3120. (a) T=4 capsid; (d) T=3 capsid. Capsid protein is blue; Fab density is pink. The capsids are viewed along a twofold symmetry axis. Control (unlabeled) capsids are shown (c and f) (from reference 15). (b and e) Lattice diagrams of single triangular facets, as marked (on panels c and f): they show the packing of dimers; the positions of symmetry axes; and the placement of quasiequivalent subunits (A, B, C, and D). Rings marking the positions of the epitopes are shaded according to occupancy: the C-D site on the T=4 capsid is dark, 100% occupancy; the A-A sites around the fivefold vertex are gray, 20 to 40% occupancy; the unoccupied D-B and B-C sites on T=4 and B-C and C-B sites on T=3 have empty rings. Bar = 50 Å.

To compare epitopes at different quasiequivalent sites, a pairof adjacent C-D subunits was aligned visually with correspondingpairs of A-A, B-C, and D-B subunits on the T=4 capsid and withpairs of A-A, B-C, and C-B subunits on the T=3 capsid. The C25-29residues were used as a primary reference in these alignments.The positions of the Fabs bound at the A=A sites on both theT=4 and T=3 capsids were assigned by applying the same rotationsand translations that moved the C and D capsid subunits to theA=A sites to the Fab overlying the C-D epitope. To allow a secondFab to bind at the same fivefold axis on the T=3 capsid, thefirst Fab was rotated a few degrees about its long axis, anda second Fab placed at an A=A site situated 144° aroundthe fivefold axis from the first site. Density was then computedaccording to procedures and programs previously described (6,20).

RESULTS

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Results

Visualization of Fab-labeled capsids. HBV capsid protein consists of a core domain (residues 1 to149) and a nucleic acid-binding protamine domain (residues 150to 183). Core domains self-assemble into capsids (7). HBV capsidsare dimorphic, the two variants having triangulation numbersof T=4 (120 icosahedrally coordinated dimers) and T=3 (90 dimers),respectively (17, 42). For the present experiments, capsidswere prepared by expressing a core domain construct in Escherichiacoli, isolating the capsids, dissociating them and further purifyingthe protein, and then reassembling capsids. The majority ofcapsids in such preparations are T=4 (44).

The immunogen used to generate MAb 3120 was HBV nucleocapsidsisolated from enveloped virions (35). We found this antibodyto be negative in Western blots (data not shown), indicatingthat it recognizes a conformational epitope (see also reference25). A positive immunoprecipitation reaction was elicited onlywith capsids, this assay being negative with dissociated butfolded dimers (data not shown).

Fab fragments were prepared and incubated with capsids. Negativestaining EM showed that the capsids were densely covered withFabs: accordingly, cryo-micrographs were also recorded. Three-dimensionaldensity maps were calculated for both capsids (Fig. 1a and dand 2a and d). The T=4 map has a resolution of $~$ 10 Å, andthe T=3 map, based on fewer particles, is at $~$ 14 Å. Fab-relateddensity (pink in Fig. 1 and 2) is associated with the surfacesof both capsids, but its distribution differs markedly betweenthem.

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FIG. 2. Interior views of cryo-EM density maps of HBV capsids labeled with Fab 3120, as represented by surface rendering (a and d) and in central sections (b and e [left half]). (a and b) T=4 capsid; (d and e) T=3 capsid. The capsids are viewed along a twofold axis of symmetry. (a and d) Capsid protein is blue, and Fab-derived density is pink. In the sections, protein density is dark. The corresponding sections from the quasi-atomic models, as limited to the same resolution as the T=4 reconstruction, are also shown (c and e [right half]). Note the close match between the cryo-EM density and the modeled density (cf. panels b and c and the left and right halves of panel e). (b) The two Fabs bound at C-D epitope sites on the T=4 capsid are marked in with white arrows. (a to e) Fivefold symmetry axes are labeled with pentagons. One example of the axial pencil of density on the T=4 fivefold axis is marked with a black arrow (b and c) or a white arrow in (a). (f) Schematic diagrams of the steric exclusion patterns at the fivefold vertices of both capsids (T=4 and T=3 A-A sites) and at the twofold axis of the T=4 capsid (T=4 C-D site) are shown. Bar = 50 Å (a to e).

Locations of Fab binding sites on the capsid surface. There are four quasiequivalent subunits on the T=4 capsid andthree on the T=3 capsid, each present in 60 copies. The T=4subunits are designated A, B, C, and D, and the T = 3 subunitsare A, B, and C (44), although the latter do not necessarilyhave the same conformations and intralattice interactions asthe similarly named T = 4 subunits. The building blocks aredimers and their placement on both surface lattices is shownschematically in Fig. 1b and e. Together, the two capsids presentseven quasiequivalent variants of any epitope.

The 25-Å-long spikes that protrude from the capsid surfaceare four-helix bundles (44) formed by two ${alpha}$ -helical hairpinsat the dimer interface (9, 15). Two copies of the so-calledimmunodominant loop, covering approximately residues 76 to 84(28), are present at the tip of each spike (14, 43). However,MAb 3120 does not bind to the spike: rather, its epitope lieson the intervening "floor" region. This property is consistentwith the observation that MAb 3120 bound to capsids in whichthe immunodominant loop was substituted with an exogenous peptide(29).

On the T=4 capsid, only one of the four quasiequivalent sitesis occupied by density corresponding in size and shape to aFab molecule (Fig. 1a, arrow). Elsewhere, and on the T=3 capsid,the observed distribution of bound density represents the superpositionof Fabs bound at adjacent sites which cannot be simultaneouslyoccupied because of steric interference (Fig. 2f). Consequently,their molecular envelopes merge in the density maps, so thatfeatures seen in surface renderings do not have the shapes ofindividual Fabs. Nevertheless, the maps are interpretable (seebelow).

The Fabs' footprints are marked on lattice diagrams in Fig.1b and 1e, shaded according to occupancy, where the darkestshades indicate full occupancy and the lightest shades indicatezero occupancy. On the T=4 capsid, the fully occupied epitopeoverlies the interface between the C and D subunits. Interestingly,there is zero occupancy of the D-B and B-C sites around thesame axis of twofold symmetry (which is also a quasi-sixfoldaxis). It follows that the avidity of Fab 3120's binding tothe C-D epitope is much higher than to the D-B and B-C epitopes,presumably reflecting local structural variations. A consequenceof this dominance by the C-D sites is that Fabs bound theredo not become merged in the density map with Fabs at the D-Band B-C sites, which are unoccupied. This serendipitous propertyfacilitated quasi-atomic modeling of the interaction betweenthe Fab and the T=4 capsid, and identification of the peptidesthat make up the epitope (see below).

Differential binding of Fabs to quasiequivalent epitopes. A thin pencil of density extends along the fivefold axis ofthe T=4 capsid (Fig. 1a and 2a and b). Although noise levelsin icosahedral reconstructions tend to be elevated close tofivefold axes, this density is at the same level as the capsidshell (representing 100% occupancy) and far exceeds the noiselevel. It is contributed by Fabs bound to the A-A sites aroundthis axis. The low value of other Fab-related density in thisregion (Fig. 2b) implies that only one of the five A-A sitesis occupied at any given vertex, where a bound Fab occludesthe other four sites slightly but enough to repel Fabs fromthem. This exclusion pattern is shown schematically in Fig.2f. Thus, the pencil of axial density represents the overlapvolume common to Fabs bound at all five sites, one of whichis randomly occupied on each vertex. Accordingly, there are $~$ 72 Fabs bound per T=4 capsid: $~$ 60 at C-D sites at $~$ 100% occupancyand $~$ 12 at A-A sites at $~$ 20% occupancy.

The T=3 capsid exhibits less binding: in particular, there iszero occupancy of any of the six potential sites (three B-Cand three C-B) around the threefold axis (cf. Fig. 1d and e).There is density associated with the A-A sites around the fivefoldaxis but it is distributed differently from the correspondingregion of the T=4 capsid (cf. Fig. 2e and b, as well as Fig.2d and a). We infer that a slight alteration in the bindingaspect of Fabs at the T=3 A-A sites allows two molecules tobind per vertex instead of one, i.e., a bound Fab occludes thetwo adjacent sites but not the sites on the other side of thefivefold axis, where a second Fab may bind (Fig. 2f). This smallchange in binding aspect results in a qualitatively distinctpattern of density in the reconstruction.

In this context, we note that the two Fabs bound at apposingC-D sites on the T=4 capsid are almost touching (Fig. 2b, whitearrows; Fig. 3b), and slight pivoting of one bound Fab towardthe twofold axis that lies between them would occlude the othersite. At the T=4 A-A sites, such impedance does occur and limitsbinding to one Fab per vertex. At the T=3 A-A sites, a slightlydifferent binding aspect allows a second Fab to attach on theother side of the same vertex. When portrayed by surface rendering(Fig. 1d), the bound Fabs produce a five-fingered ring of coalescentdensity whose fingers represent the overlap volumes of pairsof adjacent subunits (Fig. 2f). Thus, maximum occupancy of accessibleA-A sites corresponds to 24 Fabs per T=3 capsid.

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FIG. 3. Quasi-atomic model of the interaction of Fab 3120 with the T=4 capsid in the vicinity of the C-D epitope site. Atomic coordinates of a Fab (PDB—1FPT [40] shown in tan) were fitted into the cryo-EM reconstruction of the capsid-Fab 3120 complex (maroon mesh), which already housed a quasi-atomic model of the capsid: subunit A (green), B (yellow), C (red), and D (blue). An excellent fit was obtained. Capsid protein residues at the capsid-Fab interface are colored white or cyan. (a) This slab of density, showing a side-view of one Fab, contains three of the five peptide segments that make up the epitope, marked in cyan and white. (b) Face-on view of two Fabs related by a twofold symmetry axis. This slab contains four of the five the epitope peptides, marked cyan and white (see Fig. 4). The figure was produced with DINO (http://www.dino3d.org).

Characterization of the epitope. To explore the interaction of Fab 3120 with its epitope, weused our cryo-EM density maps of decorated capsids to constructquasi-atomic models. For the capsids, we used models derivedfrom the crystal structure of the T=4 capsid at 3.3 Åresolution (43) in which the dimers were positioned so as tooptimally fit our cryo-EM density maps. For the Fab, we usedfour crystal structures of molecules of the same subtype (IgG2a).The Fabs were docked into the density overlying the C-D siteby an automatic procedure (11). All four probes fitted snuglyinto the density map (e.g., Fig. 3). To test the robustnessof the fit, we calculated pairwise RMS deviations between themfor the antigen-binding domains, omitting the variable-lengthloops from these comparisons. The deviations ranged from 1.1to 1.8 Å. We conclude that although the structure of MAb3120 has not been determined, it is likely to be closely similarto the Fabs analyzed and therefore its position is specifiedby the cryo-EM map to within $~$ 1 Å in each dimension.

To further appraise the verisimilitude of the quasi-atomic modelof the Fab-labeled capsid, we excised its central section afterband-limiting the model to the same resolution as the cryo-EMmap and compared them (cf. Fig. 2c and b). An excellent matchis observed not only with respect to the capsid shell but alsowith respect to the detailed substructure of the Fabs boundat the C-D sites (white arrows, Fig. 2b). To complete the labeling,a Fab was also assigned to the interface between adjacent A-Asubunits at the same position relative to them as it occupiesrelative to the C-D interface (see Materials and Methods). Eachof the five A-A sites per vertex was labeled at 20% occupancy.This procedure generated almost perfectly the 'pencil of density'that represents the overlap region along the 5-fold axis, andalso reproduced the salient features of the faint surroundingdensity (cf. Fig. 2c and b). The same procedure was appliedat the A-A sites on the T=3 capsid, in this case rotating theFab by a few degrees and applying 40% occupancy, according tothe inferred labeling pattern (Fig. 2f). Again, this procedureclosely reproduced the Fab-associated features seen in the cryo-EMdensity map (cf. the apposing halves of Fig. 2e).

Two of the four Fab crystal structures coopted for this analysisincluded bound peptides—their respective linear epitopes.We initially thought that once the Fab-peptide complexes weredocked into the cryo-EM map, the peptides might serve as markersfor the viral epitope, but their positions vary considerablybetween the two examples. Accordingly, we focused on the regionsat which there is strongest continuity of density between Fab3120 and the capsid at the C-D site as primary indicator ofthe interaction. The portion of the density map shown in Fig.4b shows two such interactions. The modeling suggested thatthe first complementarity-determining loop on the light chainof MAb 3120 and the second complementarity-determining loopon its heavy chain are, respectively, engaged. On the basisof this analysis, we identified the capsid protein peptidesthat come closest to the Fab, concluding that five peptideson two adjacent subunits contribute to the epitope. D20-D22,D129-D132, and C25-C29 (white in Fig. 4a) are in the regionof highest interfacial density and therefore are likely to havethe strongest interactions: C126-C127 and C20-C22 (cyan in Fig.4a) are also involved, but in lower density regions. The positionsof the five peptides are marked in Fig. 4a.

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FIG. 4. (a) Model of region around a twofold axis on the HBV T=4 capsid, showing the residues that bind to Fab 3120 at the C-D interface. Subunits are colored as follows: A, green; B, yellow; C, red; D, blue. An oval marks the twofold axis. The major interactions involve peptides D20-D22, D129-D132, and C25-C29 (white), where continuity of density is seen between the capsid and the Fab when the map is contoured at 2.3 sigma above background. Secondary interactions involve C126-C127 and C20-C22 (cyan), where continuity of density is seen between the capsid and the Fab when the map is contoured at 1.8 ${sigma}$ above background. (b) Very high magnification detail of the interface region between Fab 3120 and the C-D epitope. Cryo-EM density is contoured at 2.3 ${sigma}$ (maroon mesh) with quasi-atomic models showing interaction of Fab 3120 with capsid residues C25-C29 (left) and D129-D132 (right). A Fab (tan) (PDB-1BOG; [22]), fitted into the cryo-EM density (maroon mesh), is shown along with its peptide antigen (gray). Subunits A and B and the interactions of D20-D22, C20-C22, and C126-C127 with the Fab are not seen in this view. Residues also implicated in the interaction (density = 1.8 ${sigma}$ in interface) are cyan. The figure was produced with DINO.

DISCUSSION

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Discussion

Epitopes exposed on folded proteins mainly involve surface loops.It has been argued that all such epitopes should be discontinuous,on grounds that the size of the antigen-combining surface ofa Fab is such that more than one peptide loop is required tofully engage it (4, 24). Formally, this argument is persuasivebut, in practice, many peptides are sufficiently reactive withantibodies to serve as highly specific reagents in immunochemicalassays. In such cases, although several peptides may participatein the native epitope, one of them binds the antibody stronglyenough to yield a significant interaction on its own; i.e.,it is de facto a linear epitope. On the other hand, strictlyconformational antibodies recognize only the native antigen.The present study is concerned with methods to identify thepeptides involved in epitopes of the latter kind and with exploringthe dependence of binding affinity upon conformation. Whereasconformational antibodies are supposed to bind their nativeantigens, MAb 3120 binds to only three of seven quasiequivalentvariants of its epitope.

Quasiequivalent variations in binding affinity. The epitope lies on the capsid surface, straddling the interfacebetween adjacent subunits packed around symmetry axes (Fig.1). The principal binding site involves C and D subunits onthe T=4 capsid. There are two such sites on either side of eachtwofold axis. Two more pairs of quasiequivalent sites aroundthe same axis (D-B and B-C) are unoccupied. Once the C-D sitesare occupied, access to the neighboring sites is blocked, sothe question of whether they retain some binding affinity ismoot.

On both capsids, the Fab binds to A-A sites around the fivefoldaxes but steric blocking prevents occupation of more than onesite per vertex on the T=4 capsid or two sites per vertex onthe T=3 capsid.

On the T=3 capsid, both the B-C and the C-B sites (three ofeach) around the threefold axis are unoccupied (Fig. 1e). Underthe conditions of this assay, once binding to the C-D and A-Asites was complete, there was a 3:2 molar ratio of unbound Fabsto unoccupied epitopes, or an 8.4:1 molar ratio of unbound Fabsto threefold axes on T=3 capsids (anticipating steric accessibilityof one or two Fabs per such axis). Thus, the affinities of theB-C and C-B sites must be very low. However, it is not straightforwardto interpret occupancies observed in cryo-EM experiments interms of association constants.

Variability in occupancy among quasiequivalent versions of thesame epitope appears to be quite common among viral antigens.For example, marked differences in this respect have been observedbetween the penton and hexon forms of the major capsid proteinof herpes simplex virus (36) and cucumber mosaic virus (10)and on L1, the major capsid protein of papillomavirus (8). Wesuspect that a similar mechanism may underlie these disparities,i.e., small relative shifts of the peptides that make up theepitope, between different quasiequivalent sites.

Conformational nature of the MAb 3120 epitope. According to our docking experiments, this epitope consistsof five peptides on two subunits of neighboring dimers. Thisobservation explains why MAb 3120 does not react with denaturedmonomeric capsid protein nor with folded but unassembled dimers.

We tried challenging the binding of MAb 3120 to capsids witha 10-fold molar excess of two peptides, covering residues 21to 29 and 126 to 131, respectively; together, they present allof the five shorter peptides that we infer to constitute theepitope. However, essentially no interference was registeredin a Biacore assay (data not shown), whence we conclude thatthe association constants of the peptides are too low for themto compete effectively with the composite epitope, appropriatelyconfigured on the capsid surface.

We also compared the modeled structures of the seven quasiequivalentversions of the epitope. RMS differences of $~$ 2 Å were detectedamong them, but these differences did not correlate intelligiblywith occupancy. Differences of the same order were encounteredbetween the crystal structure of the T=4 capsid and the quasi-atomicmodel, reflecting either limitations of our cryo-EM model at8-Å resolution (15) (subsequently refined from 9 Åto 8 Å resolution [J. F. Conway, unpublished results]),or that there are small but real differences between the structuresstudied, arising from the mutant used, crystal packing effects,or the preparation histories of the capsids. We conclude thatsubtle shifts in the relative positions of the five peptides—whichare below our threshold of reliable detection—accountfor the observed distinctions in Fab binding affinity.

Characterizing conformational epitopes on other antigens. The first cryo-EM investigations of capsids decorated with antibodieswere performed over ten years ago (27, 38a, 38). When feasible,molecular modeling enhances the interpretability of the resultingdensity maps (38). This area of research, recently reviewedby Smith (31), now covers numerous applications, many of whichhave addressed mechanisms of antibody-based neutralization (e.g.,see references 19, 26, and 41), while others have identifiedthe locations of specific proteins (27) or individual peptides(14, 33, 34). In the present study, the relatively high resolutionof the cryo-EM analysis (10 Å) made it possible to identifythe peptides present in a discontinuous, strictly conformational,epitope.

We are optimistic that this approach will prove useful in othersystems. In virological applications, it should help to elucidateantigenicities and thus aid in vaccine design. The conditionsfor applicability are as follows: first, a high resolution structureof the antigen—or at least the component that the antibodyof interest recognizes—should be available. Second, theantigen should be large enough to be amenable to cryo-EM analysis,i.e., at least 250 kDa or so (5) (all capsids qualify). Fabbinding facilitates such analysis because it augments the sizeand irregularity of the structure to be determined. A thirdconsideration is the quality of the cryo-EM map. Although muchthe same conclusion with respect to epitope placement couldhave been reached at lower resolution, the 10-Å resolutionof our T=4 map made it possible to specify the interactionsat the Fab-capsid interface. In summary, the signal-to-noiseratio and occupancy should be high enough to clearly delineatethe Fab; the resolution should suffice to specify its orientationrelative to the point of contact; and the hand of the EM mapshould have been established.

One potential complication with antigens that have many copiesof the same epitope is steric interference between neighboringbinding sites, which results in the distribution of bound densityhaving the shape not of a single Fab but rather, of an amalgamof overlapping Fabs—e.g., see references 8 and 10 andour A-A sites in Fig. 1. However, this problem may be tackledby first identifying the footprint of the Fab on the capsidsurface and then pivoting the model structure about this contactpoint to optimize the fit in the proximal portion of the densitymap (D. M. Belnap et al., unpublished data).

ACKNOWLEDGMENTS

We thank D. Milich (Scripps) and R. Tedder and B. Ferns (MiddlesexHospital) for helpful correspondence, P. Ober (NIAMS) for programminghelp, W. Wriggers (Scripps) for help with Colores, and B. Heymann(LSBR) for software.

FOOTNOTES

* Corresponding author. Mailing address: Bldg. 50, Rm. 1517, MSC 8025, National Institutes of Health, Bethesda, MD 20892-8025. Phone: (301) 496-0132. Fax: (301) 480-1191. E-mail: Alasdair_Steven{at}nih.gov.

REFERENCES

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