Structural Basis of Neutralization by a Human Anti-severe Acute Respiratory Syndrome Spike Protein Antibody, 80R

Abstract

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 Å resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 Å resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity. Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 Å resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 Å resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity. Severe acute respiratory syndrome (SARS), 3The abbreviations used are: SARS, severe acute respiratory syndrome; CoV, coronavirus; ACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain; CDR, complementarity-determining region. a newly emerged infectious disease, claimed 813 lives from ∼8000 patients during a 2003 global epidemic. In severe illness, influenza-like symptoms quickly progress to pneumonia, hypoxia, and acute respiratory distress and failure, resulting in 10% overall death rate with exceptionally high mortality among the elderly (1Donnelly C.A. Ghani A.C. Leung G.M. Hedley A.J. Fraser C. Riley S. Abu-Raddad L.J. Ho L.M. Thach T.Q. Chau P. Chan K.P. Lam T.H. Tse L.Y. Tsang T. Liu S.H. Kong J.H. Lau E.M. Ferguson N.M. Anderson R.M. Lancet. 2003; 361: 1761-1766Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar). A novel coronavirus (SARS-CoV) has been identified as the etiological agent of SARS. The SARS-CoV surface spike protein S mediates viral entry into the host cell (2Holmes K.V. J. Clin. Investig. 2003; 111: 1605-1609Crossref PubMed Scopus (199) Google Scholar) and includes two functional domains as follows: S1 (Gly13-Arg667) and S2 (Ser668-Thr1255). S1 contains the host-specific receptor binding domain (RBD), whereas S2 mediates fusion between viral and host cell membranes (3Xu Y. Lou Z. Liu Y. Pang H. Tien P. Gao G.F. Rao Z. J. Biol. Chem. 2004; 279: 49414-49419Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for the SARS-CoV (4Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google Scholar). The recently determined structure of the S1-RBD in complex with the extracellular domain of ACE2 (5Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar) illustrates the structural basis for the initial step of virus-host recognition. As the mediator of host-specific SARS infection and a major viral surface antigen, the S protein is an attractive candidate for both vaccine development and immunotherapy. Marasco and co-workers (6Sui J. Li W. Murakami A. Tamin A. Matthews L.J. Wong S.K. Moore M.J. Tallarico A.S. Olurinde M. Choe H. Anderson L.J. Bellini W.J. Farzan M. Marasco W.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2536-2541Crossref PubMed Scopus (485) Google Scholar) previously identified a potent neutralizing human monoclonal antibody against the S1 RBD, designated “80R,” from two nonimmune (i.e. not restricted by B cell recombination) human antibody libraries. 80R binds S1 with nanomolar affinity, blocks the binding of S1 to ACE2, prevents the formation of syncytia in vitro (6Sui J. Li W. Murakami A. Tamin A. Matthews L.J. Wong S.K. Moore M.J. Tallarico A.S. Olurinde M. Choe H. Anderson L.J. Bellini W.J. Farzan M. Marasco W.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2536-2541Crossref PubMed Scopus (485) Google Scholar), and inhibits viral replication in vivo (7Sui J. Li W. Roberts A. Matthews L.J. Murakami A. Vogel L. Wong S.K. Subbarao K. Farzan M. Marasco W.A. J. Virol. 2005; 79: 5900-5906Crossref PubMed Scopus (134) Google Scholar). Deletion studies have shown that the 80R epitope on S1 is located in the minimal ACE2 binding domain, between residues 324 and 503 (6Sui J. Li W. Murakami A. Tamin A. Matthews L.J. Wong S.K. Moore M.J. Tallarico A.S. Olurinde M. Choe H. Anderson L.J. Bellini W.J. Farzan M. Marasco W.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2536-2541Crossref PubMed Scopus (485) Google Scholar, 7Sui J. Li W. Roberts A. Matthews L.J. Murakami A. Vogel L. Wong S.K. Subbarao K. Farzan M. Marasco W.A. J. Virol. 2005; 79: 5900-5906Crossref PubMed Scopus (134) Google Scholar). Here, we report the crystal structure of the S1-RBD both alone and in complex with 80R. The complex structure reveals the basis of the broad neutralizing ability of 80R and will facilitate the design of immunotherapeutics in the case of a future SARS outbreak. We further show that the S1-RBD forms dimers by means of an unexpected reorganization of the region distal to the receptor-binding surface. The dimers are disrupted by complex formation, and we discuss the possibility that receptor binding plays an active role in the initial steps of viral uncoating. Protein Expression, Purification, and Crystallization—The gene encoding single chain (VH-linker-VL) antibody 80R (scFv) was cloned into pET22b (Novagen) containing an N-terminal periplasmic secretion signal pelB, and a thrombin-removable C-terminal His6 tag. 80R was overexpressed in BL21(DE3) cells at 30 °C for 15 h with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Protein was purified by HisBind nickel-nitrilotriacetic acid (Novagen) column and Superdex 200 gel filtration chromatography (Amersham Biosciences) after thrombin digestion. The gene encoding S1-RBD (residues 318-510) was cloned into vector pAcGP67A (Pharmingen) containing an N-terminal gp67 secretion signal and a thrombin-cleavable C-terminal His6 tag. It was expressed in Sf9 cells (Invitrogen) with a multiplicity of infection = 5 for 72 h. Similar to 80R, S1-RBD was purified from the media with HisBind nickel-nitrilotriacetic acid and Superdex 200 columns, with thrombin digestion. N-Linked glycosylation was removed by incubation with peptide:N-glycosidase F (New England Biolabs) at 23 °C, as monitored by SDS-PAGE. S1 RBD-80R complexes were formed by mixing the two purified components and isolated by gel filtration with Superdex 200 in 10 mm Tris-HCl, 150 mm NaCl, pH 7.4. Peak fractions were pooled and concentrated to ∼7 mg/ml. For S1-RBD crystal growth, the protein was also concentrated to ∼7 mg/ml. Crystals grew by the hanging drop vapor diffusion method at 17 °C over ∼21 days. For S1-RBD, 2 μl of S1-RBD was mixed with an equal volume of well solution containing 4% w/v polyethylene glycol 4000, 0.1 m sodium acetate, pH 4.6. For the S1-RBD-80R complex, 2 μl of the complex was mixed with an equal volume of well solution containing 12.5% w/v polyethylene glycol 4000, 0.1 m sodium acetate, 0.2 m ammonium sulfate, pH 4.6. Data Collection, Structure Determination, and Refinement— X-ray diffraction data were collected at the National Synchrotron Light Source beamline X6A and X29A for S1-RBD crystals, the Stanford Synchrotron Radiation Laboratory beamline 11.1, and at the Advanced Light Source beamlines 5.0.3 and 12.3.1 for crystals of the S1-RBD-80R complex. Glycerol (25%) was used as a cryoprotectant in both cases. All the data were processed with DENZO and SCALEPACK or with the HKL2000 package (8Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). Crystals of S1 RBD adopt space group P43212 with unit cell dimensions a = 75.9 and c = 235.8 (Table 1).TABLE 1Data collection and refinement statisticsS1-RBDS1-RBD-80RData collectionCell parametersa = 75.9, c = 235.9 Åa = 47.5, b = 175.9, c = 67.6 Å; β = 96.6°Space groupP43212P21Resolution (Å)2.22.3Total reflections233011159047Unique reflections3603651915Completeness (%)aNumbers in parentheses correspond to the highest resolution shell (2.28-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)99.9 (99.9)93.8 (87.0)Average I/σ(I)aNumbers in parentheses correspond to the highest resolution shell (2.28-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)24.7 (2.0)8.8 (1.9)RmergeaNumbers in parentheses correspond to the highest resolution shell (2.28-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)0.098 (0.739)0.145 (0.571)Redundancy6.53.1RefinementRworkbNumbers in parentheses correspond to the highest resolution shell (2.26-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)0.182 (0.230)0.248 (0.301)Rfree (5% data)bNumbers in parentheses correspond to the highest resolution shell (2.26-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)0.213 (0.289)0.295 (0.391)r.m.s.d. bond distance (Å)cr.m.s.d., root meant square deviation0.0130.009r.m.s.d. bond angle (°)1.491.22Average B value50.037.1Solvent atoms152470Ramachandran plotResidues in most favored regions276631Residues in additional allowed regions3581Residues in generously allowed regions35Residues in disallowed regions00a Numbers in parentheses correspond to the highest resolution shell (2.28-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)b Numbers in parentheses correspond to the highest resolution shell (2.26-2.20 Å for S1 RBD; 2.29-2.38 Å for S1 RBD-80R)c r.m.s.d., root meant square deviation Open table in a new tab Crystals of the S1-RBD-80R complex adopt space group P21 with unit cell dimensions a = 47.5, b = 175.9, c = 67.6, β = 96.6°. The crystals display a lattice-translocation defect in which a fraction of the layers have a translational offset, resulting in periodic sharp and diffuse rows of reflections (Fig. 1). Similar defects were first described by Bragg and Howells (9Bragg W.L. Howells E.R. Acta Crystallogr. 1954; 7: 409-411Crossref Google Scholar). Different crystals displayed different degrees of lattice defects, and data merged poorly between crystals. By using a single crystal we were able to collect a data set of good quality with a final RMERGE = 0.145 and completeness of 93.8% to 2.3 Å resolution. Processing the data required careful optimization of integration profiles and the imposition of a fixed mosaicity (0.45°). Correlation between the offset layers caused the appearance of a strong off-origin peak (65% of the origin) in the native Patterson map at (1/3, 0, 0), indicating that the dislocation occurred along the a* direction. Additional features of the Patterson map were visible at ∼1/10 of the origin peak and provided a measure of the severity of the defect among different crystals. The averaged intensity for the layers of reflections showed a periodic variation that corresponded to the sharp and diffuse layers, and we used the procedure developed by Wang et al. (10Wang J. Kamtekar S. Berman A.J. Steitz T.A. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2005; 61: 67-74Crossref PubMed Scopus (34) Google Scholar) to correct for the intensity modulation (Fig. 2). We calculated average intensities for individual h layers, and applied a correction to the intensities using Equation 1, ICOR=IMEAS/(A+B cos(2πhΔx))(Eq. 1) where A and B were obtained by least square fitting of the averaged measured intensities. The ratio of the parameters B and A (B/A = 0.65) coincided with the height ratio of the Patterson peak at (1/3, 0, 0), as required by the lattice-translocation theory presented by Wang. The corrected intensity distribution (Fig. 2b) was used for the structure solution and the refinement.FIGURE 2h layer intensities before and after correction. a, the lattice defect results in a strong-weak-weak pattern of intensities along h, which were corrected (b) according to the procedure of Wang et al. (10Wang J. Kamtekar S. Berman A.J. Steitz T.A. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2005; 61: 67-74Crossref PubMed Scopus (34) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The structure of the S1-RBD-80R complex was determined using the Joint Center for Structural Genomics molecular replacement pipeline (11Schwarzenbacher R. Godzik A. Grzechnik S.K. Jaroszewski L. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: 1229-1236Crossref PubMed Scopus (152) Google Scholar), which employs a modified version of MOLREP (12Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar), and independently using PHASER (13McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1599) Google Scholar), with the S1-RBD domain from the S1-RBD-ACE2 complex and the scFv domain from the scFv-turkey egg-white lysozyme complex (Protein Data Bank code 1DZB) as The unit contains two of S1 The final includes residues 1) and of S1 RBD and residues 1) and of 80R, and was for the from molecular replacement were to of refinement with the Acta Crystallogr. Sect. D. Biol. Crystallogr. 1997; PubMed Scopus Google Scholar) with in G.M. W.L. P. Grosse-Kunstleve R.W. J. M. Read R.J. L.M. T. Acta Crystallogr. Sect. D. Biol. Crystallogr. PubMed Scopus Google Scholar) and with T.A. M. Acta Crystallogr. Sect. A. PubMed Scopus Google Scholar) and P. K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: PubMed Scopus Google Scholar). The structure of uncomplexed S1-RBD showed lattice was determined by molecular replacement with PHASER (13McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1599) Google Scholar) using S1-RBD from the structure of the S1-RBD-ACE2 complex (Protein Data Bank code as the The unit contains two of S1-RBD as a The final includes residues of both and parameters are as with R.J. J. Appl. Crystallogr. Google Scholar) (Table 1). are and for the uncomplexed S1-RBD and the S1-RBD-80R complex, The for the S1 RBD-80R complex by the of the lattice defect and the integration of as previously (10Wang J. Kamtekar S. Berman A.J. Steitz T.A. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2005; 61: 67-74Crossref PubMed Scopus (34) Google Scholar). the final map for the S1 RBD-80R complex is of quality (Fig. and the is for most of the residues at 2.3 Å resolution. have been in the Protein Data Bank with and Structure of the S1-RBD-80R determined the crystal structure of the S1-RBD-80R complex at 2.3 Å resolution and and 1). The S1 RBD has a very structure to that in the ACE2 complex (Fig. The complex interface antibody complementarity-determining region which into the surface on the S1 receptor-binding et al. C. J. Biol. PubMed Scopus Google Scholar, C. A. M. S. D. Nature. PubMed Scopus Google Scholar) showed that a of chain for 5 of the which is and in and that from acid For the of 80R, and adopt chain to by the is to that of the antibody (Protein Data Bank code The is and well A that is of the and (Fig. and also plays a major role in the the (5Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar) of S1 residues between 80R and S1 RBD 80R residues are on the and or region S1 residues in with 80R residues are in subsequent and S1 for 5 residues and which are at the residues and are at the Open table in a new tab ACE2 employs a different by a that the surface of the 80R epitope on S1 overlaps very closely with the ACE2-binding surface (Fig. of the residues and on S1 that 80R, 17 of also in the The interface of protein with for The a measure of S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), is for the that of The surface and volume provide a rationale for the binding and neutralizing of the antibody. The structure a rationale for is in both and mutations both 80R binding and ACE2 binding (7Sui J. Li W. Roberts A. Matthews L.J. Murakami A. Vogel L. Wong S.K. Subbarao K. Farzan M. Marasco W.A. J. Virol. 2005; 79: 5900-5906Crossref PubMed Scopus (134) Google Scholar, W. C. Sui J. J.H. Moore M.J. S. Wong S.K. K. Vasilieva N. Murakami A. Y. Marasco W.A. Y. Choe H. Farzan M. J. 2005; PubMed Scopus Google further that antibody binding, at and are not in the by the of residues in a that the binding interface to the S1 RBD and the that the (Fig. between the two in the role of S1 or mutations binding to 80R have on ACE2 binding (7Sui J. Li W. Roberts A. Matthews L.J. Murakami A. Vogel L. Wong S.K. Subbarao K. Farzan M. Marasco W.A. J. Virol. 2005; 79: 5900-5906Crossref PubMed Scopus (134) Google Scholar). at the of the an to 1) and an to of 80R, where as in the complex. of S1 RBD to ACE2 or 80R is of glycosylation (6Sui J. Li W. Murakami A. Tamin A. Matthews L.J. Wong S.K. Moore M.J. Tallarico A.S. Olurinde M. Choe H. Anderson L.J. Bellini W.J. Farzan M. Marasco W.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2536-2541Crossref PubMed Scopus (485) Google Scholar, S. P. Virol. J. 2005; PubMed Scopus Google and in the S1 RBD are from the binding Structure of the also determined the crystal structure of the uncomplexed S1-RBD (residues 318-510) at 2.2 Å resolution 5 and and 1). with structure in complex with 80R or ACE2, the receptor-binding including the is to structure in the are extensive and of the region distal to the surface (Fig. which to the formation of an extensive dimer interface with a surface of (Fig. The major reorganization in structural structure as in F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar) as follows: the between 2 and containing B that the new B is and to in the B from the the dimer A to by Å to a new to the and the also a The dimer is formed by the of the and from and is in of the S1-RBD a, S1 are in and by a The receptor binding and are as in a by a to show the molecular of the S1-RBD dimer with two of ACE2 The is a with a in to the in the dimer A antibody dimers on the viral the is for binding both on a single dimer Large Image Figure ViewerDownload Hi-res image Download (PPT) dimer is by the to and to have a binding with the and In with the structural gel filtration studies of S1 RBD a in solution at not has been that the S1 domain also as a dimer J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of S1 RBD on the surface of the dimers (Fig. with to the S2 the surface of the dimer two from into distance = not a We propose a in which the S1 dimers two receptor-binding from the viral membrane surface. A role for the S1 dimers is to S protein via S2 on the viral surface Y. W. Wang C. J. J. S. 2004; Google Scholar), to the structural of the two ACE2 the S1 dimer to between the (Fig. which S1 is in crystals of the complex. in the complex, S1 dimers and in case the two residues in the are with to in the uncomplexed S1 and the interface is In of two 80R the uncomplexed S1 dimer not to and in case the dimer is by lattice the that binding of in vivo of S protein in a S for subsequent membrane fusion events by the S2 A role for viral is well in the J. Virol. 2003; PubMed Scopus Google Scholar), and has also been described for the protein of virus W. S. Full Text Full Text PDF PubMed Scopus Google Scholar). further are required to for and co-workers W. C. Sui J. J.H. Moore M.J. S. Wong S.K. K. Vasilieva N. Murakami A. Y. Marasco W.A. Y. Choe H. Farzan M. J. 2005; PubMed Scopus Google Scholar) previously that 80R and that during the outbreak. the 80R epitope on S1 overlaps closely with the ACE2-binding site, we for most residues on S1 at the binding site, on S that 80R is to binding to ACE2 as A is the which was in the from during the were for an that in the infection of patients in a Wang K. L.C. Gao H. F. H. Liu Li W.J. Anderson L. Wang M. Gao Y. M. L. L.Y. Li H. F. L.J. S. Kong L. P. H. A. A. A. N. Li Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). The 80R antibody not mutants, as were also and of were By the and profiles of newly S1 of the neutralizing epitope of 80R, which we have in an with 80R should in a future SARS outbreak. In of 80R provide for the resulting in virus and for of the We and J. at beamline H. and at beamline X29A Synchrotron Light and K. at beamlines 12.3.1 and C. at 5.0.3 Light for with data and C. and L. for Download with