Michael A. DiMattia, Norman R. Watts, Stephen J. Stahl, Jonathan M. Grimes, Alasdair C. Steven, David I. Stuart, and Paul T.

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1 Structure, Volume 21 Supplemental Information Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein Michael A. DiMattia, Norman R. Watts, Stephen J. Stahl, Jonathan M. Grimes, Alasdair C. Steven, David I. Stuart, and Paul T. Wingfield Inventory of Supplemental Information Supplemental Data: four Figures The figures provide additional views of the HBeAg antigenic structure depicting the epitopes of various antibodies (including e6), as well as the electron density for HBeAg. Figure S1 Crystalline lattice of HBeAg-Fab e6 complex, Electron density map (2Fo-Fc) of region of HBeAg, and B-factor distribution, related to Figure 1. Figure S2 Sequence alignment of Hepadnaviridae HBeAg sequences, related to Figure 2. Figure S3 The epitopes of additional anti-hbc/eag antibodies are mapped onto HBeAg, related to Figure 4. Figure S4 Structural comparison of Fab e6 epitope (on HBeAg) and dimer-dimer capsid interface (on HBcAg), related to Figure 4. Supplemental Experimental Procedures Additional experimental methods provide sequence information for proteins involved in this study, experimental procedures of e6 Fab sequencing, purification and crystallization of all proteins involved.

2 SUPPLEMENTAL DATA A B C Figure S1. Structure validation, related to Figure 1. (A) Electron density map (2Fo-Fc) of region of HBeAg. Left panel depicts front view, as blue mesh and contoured at 1.0σ. The backbone ribbon is colored as in Figure 1. Right panel has the same representation, but 60 rotated to show the density for α-helix 2 and the propeptide. (B) Crystalline lattice of HBeAg-Fab e6 complex. HBeAg depicted in ribbon (orange and yellow) and e6 Fabs are shown in shades of green. The lattice arrangement explains how the Fab aided crystallization of HBeAg, where previously the molecule had been prone to aggregation. All of the crystal contacts are mediated by the Fabs. (C) B-factor distribution of HBeAg-Fab e6 crystal structure asymmetric unit colored by B-factor per residue, ranging from navy blue (low) to red (high). The B-factors reflects the distribution of thermal energy in the molecules, and specifically show that HBeAg is more thermally active than Fab, particularly at spike apex. The minimal contact region between Fabs may add steric strain that further destabilizes the spike apex.

3 Figure S2. Sequence alignment of Hepadnaviridae HBeAg sequences, related to Figure 2. The top eight represent Orthoviridae (mammalian) and the remaining five encompass members of Avihepadnaviridae (birds). Secondary structure elements are colored as in Fig. 1 and propeptide sequences are boxed in magenta.

4 Figure S3. The epitopes of additional anti-hbc/eag antibodies mapped onto HBeAg, related to Figure 4. The epitopes of antibodies 9c8 and 842 splay apart into two disparate sub-epitopes, similar to those of F11A4 and The epitopes of antibodies 88 and 312, however, only bind one helical hairpin of the capsid spike.

5 Figure S4. Structural comparison of Fab e6 epitope (on HBeAg) and dimer-dimer capsid interface (on HBcAg), related to Figure 4. (A) e6 epitope mapped onto HBeAg monomer. Most deeply buried residues are depicted in hot pink. (B) One of two inter-dimer interfaces of HBcAg capsids, most deeply buried residues in dark blue.

6 SUPPLEMENTAL EXPERIMENTAL PROCEDURES HBeAg sequence SKLCLGWLWGMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTAAALYRDALESPEHASPHHTA LRQAILCWGDLMTLATWVGTNLEDPASRDLVVSYVNTNVGLKFRQLLWFHISALTFGRETVLEY LVSFAVWIRTPPAYRPPNAPILSTLPETTVV Fab e6 light chain sequence NIMMTQSPSSLAVSAGEKVTMNCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRES GVPDRFTGSGSGTDFTLTISSVQTEDLAVYYCHQYLSSYMYTFGGGTKLEIKRADAAPTVSIFP PSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERHNSYTCEATHKTSTSPIVKSFNRN Fab e6 heavy chain sequence EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGNYIYYPD TVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTREGAYSGSSSYPMDYWGQGTSVTVSSA KTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTM SSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSG Protein Preparation We used the construct Cp(-10)149, C48A, C107A, and refer to it here as HBeAg although it differs from wildtype HBeAg in having two Ala substitutions at C48 and C107, which do not form disulfide bridges (Wingfield et al., 1995). In brief, the expressed protein was extracted from E. coli with 3 M urea, 100 mm sodium bicarbonate, ph 9.6 and subjected to gel filtration on a Superdex 200 column in the same buffer plus 2 M urea. The eag pool was further fractionated by anion exchange chromatography on Sepharose Q in 50 mm Tris-HCl, ph 8.0 (0-0.5M NaCl

7 gradient). Finally, the protein was rechromatographed on Superdex 200, ph 9.6 buffer (minus urea). This material was dimeric (~36,000 Da) as determined by analytical ultracentrifugation. Analyzed by SDS-PAGE, the protein was monomeric with a lower apparent molecular weight under non-reducing versus reducing conditions, consistent with the presence of an intramolecular disulfide C(-7) C61 (Watts et al., 2011). Fab e6 and the HBeAg-Fab e6 complex were produced as described (Watts et al., 2010). The components were mixed at an expected stoichiometry of 1:2, and excess Fab was removed by size-exclusion chromatography. The HBeAg used for crystallization included the aforementioned mutations plus G123A (Watts et al., 2011). Sequencing of e6 Fab The protein sequence of e6 V L and V H regions were determined using RT PCR (Qiagen) to sequence the mrna encoding the mouse e6 mab. Using previously reported primers (Morrison, 2002), we were able to ascertain the coding sequence for the V H region of Fab e6, but not that of the V L region. The amino acid sequence of the N-terminus of V L was determined by Alphalyse Inc. (Palo Alto, CA, 94306) to be NIMMTQSP and we synthesized a RT PCR primer for the 5 end of the V L coding region based on this amino acid sequence, which fully and exclusively matches mouse V gene IGKV8-27 ( We then synthesized a second oligonucleotide primer matching the 5 end of the secretion sequence associated with gene IGKV8-27. Both of these oligonucleotides primed DNA synthesis from the mouse mrna preparation and enabled us to obtain the coding sequence of the entire V L region. Crystallization and data collection Crystallization trials for the complex were performed using the sitting drop vapor diffusion method at a protein concentration of 5.3 mg ml -1 in 20 mm HEPES ph 8.0 at 21 C. Sitting drops were formed by mixing 100 nl of protein solution and 100 nl of reservoir solution (Walter et al.,

8 2005). Plate-shaped crystals (dimensions approximately 70 m 80 m 30 m) appeared in 5 days and grew to full size within 10 days with reservoir solution containing 20% PEG 6000 and 100 mm bicine ph 9.0. Rod-like crystals of Fab e6 (dimensions approximately 230 m 40 m 40 m) appeared in ~3 months using reservoir solution containing 20% PEG 3350, 100 mm bis-tris propane ph 6.5, and 200 mm potassium thiocyanate. Fab e6 crystals serendipitously grew from a preparation of the HBeAg-Fab e6 complex in which excess Fab was not removed after complex formation. Both HBeAg-Fab e6 and Fab e6 crystals were cryo-protected in 25% glycerol, frozen in liquid nitrogen, and screened using synchrotron radiation at the Diamond Light Source, Beamline I24, Didcot, UK. Diffraction data for the complex and Fab e6 were collected from single crystals to a minimum Bragg spacing of 3.3 Å and 2.5 Å resolution, respectively. Diffraction data were integrated and scaled using xia2 (Winter, 2010). Structure solution and refinement Initial phase information for the Fab e6 data were obtained via an automated Phaser (1994) molecular replacement (MR) search using a library of 334 Fab structures as search models that cover the full range of Fab elbow angles observed in the PDB (Stanfield et al., 2006). The correct solution was found using the structure of a monoclonal Fab fragment raised against HIV- 1 protease (PDB ID 2HRP), which shares 77% sequence identity with Fab e6. The space group is C2 with 4 Fab molecules in the asymmetric unit. The MR solution was rigid body-refined with each Ig domain treated separately, hyper-variable regions deleted, and backbone converted to poly-alanine. Positional, TLS, and individual isotropic B-factor refinement were carried out in BUSTER (Bricogne et al., 2011) using fourfold NCS restraints, followed by iterative rebuilding of the hyper-variable loops and correction of the sequence according to the separately determined Fab e6 light and heavy chains. This Fab e6 structure, refined against 2.5 Å data, was used as an MR model to obtain initial phase estimates for the HBeAg-Fab e6 data. Two Fab e6

9 molecules were placed in the P1 unit cell and refinement used local structure similarity restraints (LSSR) with the 2.5 Å resolution Fab structure as a target function (Smart et al., 2008). The HBeAg subunits were placed by first building three α-helices into the tube-like density regions in the map adjacent to each Fab e6. An HBcAg monomer (from 1QGT) was unambiguously superposed on to each α-helical framework, which provided the initial orientation of the dimer subunits. Additional electron density was clearly observed extended from C61 to near the N- terminus of the HBcAg model. Positional, group B-factor (one group per residue), and TLS refinement were used for structural refinement, with twofold NCS-restraints and restraints to the Fab structure applied to mitigate the limited resolution of the data. The Molprobity server (Chen et al., 2010) and validation tools in Coot (Emsley et al., 2010) informed the quality of the structure refinement process for both Fab e6 and HBeAg-Fab e6 complex. Refinement statistics are given in Table 1, and final refined coordinates and structure factors have been deposited for Fab e6 and HBeAg-Fab e6 with the PDB with accession codes 3V6F and 3V6Z, respectively. Structure analysis PISA interface web server was utilized for buried surface area and interacting residue analysis of the HBeAg dimer interface and the HBeAg-Fab e6 epitope-paratope interface (Krissinel and Henrick, 2007). The inter-axial angle between two HBeAg subunits was determined manually by calculating the dot product of the vectors parallel to the central helical hairpin of each subunit. The Rapido server was used to determine structurally similar sub-domains within HBeAg and HBcAg monomers (Mosca et al., 2008). A structurally similar region between the antigens was found and used for all superpositions between HBcAg and HBeAg (residues 5-34, 49-65, ). Secondary structure assignment of HBeAg was done using DSSP (Kabsch and Sander, 1983) and Stride (Heinig and Frishman, 2004). Molecular graphics were produced using Pymol (DeLano Scientific LLC).

10 Sedimentation velocity analysis and negative-stain electron microscopy HBeAg was dialyzed against phosphate buffered saline (PBS) ph 7.2, plus 300 mm NaCl (total NaCl 450 mm). A sample treated with 10 mm DTT was also dialyzed against the same buffer, plus 2 mm DTT. Under either of these conditions, dimeric HBcAg (Cp149) readily and efficiently forms capsids. Following dialysis for ~24h, samples were analyzed by sedimentation velocity. Measurement of the height (UV absorbance) of the sedimenting boundaries allows the concentrations of the various species to be determined. The oxidized and reduced HBeAg samples, were also applied to glow-discharged, poly-lysine coated carbon grids at a concentration of ~0.25 mg/ml, stained with 1% uranyl acetate and observed at x35,000 magnification in a Philips CM-120 electron microscope.