Structure of Severe Acute Respiratory Syndrome Coronavirus Receptor-binding Domain Complexed with Neutralizing Antibody
2006; Elsevier BV; Volume: 281; Issue: 23 Linguagem: Inglês
10.1074/jbc.m600697200
ISSN1083-351X
AutoresPonraj Prabakaran, Jianhua Gan, Yang Feng, Zhongyu Zhu, Vidita Choudhry, Xiaodong Xiao, Xinhua Ji, Dimiter S. Dimitrov,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoThe severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV), which caused a world-wide epidemic in 2002 and 2003, binds to a receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) of its envelope (spike, S) glycoprotein. The RBD is very immunogenic; it is a major SCV neutralization determinant and can elicit potent neutralizing antibodies capable of out-competing ACE2. However, the structural basis of RBD immunogenicity, RBD-mediated neutralization, and the role of RBD in entry steps following its binding to ACE2 have not been elucidated. By mimicking immune responses with the use of RBD as an antigen to screen a large human antibody library derived from healthy volunteers, we identified a novel potent cross-reactive SCV-neutralizing monoclonal antibody, m396, which competes with ACE2 for binding to RBD, and determined the crystal structure of the RBD-antibody complex at 2.3-Ä resolution. The antibody-bound RBD structure is completely defined, revealing two previously unresolved segments (residues 376–381 and 503–512) and a new disulfide bond (between residues 378 and 511). Interestingly, the overall structure of the m396-bound RBD is not significantly different from that of the ACE2-bound RBD. The antibody epitope is dominated by a 10-residue-long protruding β6–β7 loop with two putative ACE2-binding hotspot residues (Ile-489 and Tyr-491). These results provide a structural rationale for the function of a major determinant of SCV immunogenicity and neutralization, the development of SCV therapeutics based on the antibody paratope and epitope, and a retrovaccinology approach for the design of anti-SCV vaccines. The available structural information indicates that the SCV entry may not be mediated by ACE2-induced conformational changes in the RBD but may involve other conformational changes or/and yet to be identified coreceptors. The severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV), which caused a world-wide epidemic in 2002 and 2003, binds to a receptor, angiotensin-converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) of its envelope (spike, S) glycoprotein. The RBD is very immunogenic; it is a major SCV neutralization determinant and can elicit potent neutralizing antibodies capable of out-competing ACE2. However, the structural basis of RBD immunogenicity, RBD-mediated neutralization, and the role of RBD in entry steps following its binding to ACE2 have not been elucidated. By mimicking immune responses with the use of RBD as an antigen to screen a large human antibody library derived from healthy volunteers, we identified a novel potent cross-reactive SCV-neutralizing monoclonal antibody, m396, which competes with ACE2 for binding to RBD, and determined the crystal structure of the RBD-antibody complex at 2.3-Ä resolution. The antibody-bound RBD structure is completely defined, revealing two previously unresolved segments (residues 376–381 and 503–512) and a new disulfide bond (between residues 378 and 511). Interestingly, the overall structure of the m396-bound RBD is not significantly different from that of the ACE2-bound RBD. The antibody epitope is dominated by a 10-residue-long protruding β6–β7 loop with two putative ACE2-binding hotspot residues (Ile-489 and Tyr-491). These results provide a structural rationale for the function of a major determinant of SCV immunogenicity and neutralization, the development of SCV therapeutics based on the antibody paratope and epitope, and a retrovaccinology approach for the design of anti-SCV vaccines. The available structural information indicates that the SCV entry may not be mediated by ACE2-induced conformational changes in the RBD but may involve other conformational changes or/and yet to be identified coreceptors. The severe acute respiratory syndrome coronavirus (SARS-CoV, or SCV) 4The abbreviations used are: SCV, severe acute respiratory syndrome coronavirus (or SARS-CoV); mAb, monoclonal antibody; ACE2, angiotensin-converting enzyme 2; RBD, receptor-binding domain; Env, virus envelope glycoprotein; HIV, human immunodeficiency virus; MES, 4-morpholineethanesulfonic acid; CDR, complementarity-determining region; S glycoprotein; spike glycoprotein. infected more than 8000 humans with a fatality rate of ∼10% (1Ksiazek T.G. Erdman D. Goldsmith C.S. Zaki S.R. Peret T. Emery S. Tong S. Urbani C. Comer J.A. Lim W. Rollin P.E. Dowell S.F. Ling A.E. Humphrey C.D. Shieh W.J. Guarner J. Paddock C.D. Rota P. Fields B. DeRisi J. Yang J.Y. Cox N. Hughes J.M. LeDuc J.W. Bellini W.J. Anderson L.J. N. Engl. J. Med. 2003; 348: 1953-1966Crossref PubMed Scopus (3375) Google Scholar, 2Peiris J.S. Lai S.T. Poon L.L. Guan Y. Yam L.Y. Lim W. Nicholls J. Yee W.K. Yan W.W. Cheung M.T. Cheng V.C. Chan K.H. Tsang D.N. Yung R.W. Ng T.K. Yuen K.Y. Lancet. 2003; 361: 1319-1325Abstract Full Text Full Text PDF PubMed Scopus (2361) Google Scholar, 3Drosten C. Gunther S. Preiser W. van der W.S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. Cinatl J. Eickmann M. Escriou N. Grywna K. Kramme S. Manuguerra J.C. Muller S. Rickerts V. Sturmer M. Vieth S. Klenk H.D. Osterhaus A.D. Schmitz H. Doerr H.W. N. Engl. J. Med. 2003; 348: 1967-1976Crossref PubMed Scopus (3455) Google Scholar, 4Holmes K.V. N. Engl. J. Med. 2003; 348: 1948-1951Crossref PubMed Scopus (272) Google Scholar). Although there have been no recent outbreaks, the need to develop potent therapeutics and vaccines against a re-emerging SCV or a related virus remains of high priority. The amazingly rapid pace of SARS research for the last few years has resulted in a wealth of information for the virus, especially about its interactions with the host leading to disease and immune responses, which could also be helpful for the development of strategies to cope with other viral pathogens including influenza and HIV. Entry of viruses into animal cells is initiated by binding to cell-surface-associated receptors and can be prevented by neutralizing antibodies (nAbs) targeting the virus receptor-binding site (5Dimitrov D.S. Nat. Rev. Microbiol. 2004; 2: 109-122Crossref PubMed Scopus (393) Google Scholar, 6Smith A.E. Helenius A. Science. 2004; 304: 237-242Crossref PubMed Scopus (622) Google Scholar). In the case of SCV entry, the spike (S) glycoprotein (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 8Xiao X. Dimitrov D.S. Cell Mol. Life Sci. 2004; 61: 2428-2430Crossref PubMed Scopus (26) Google Scholar) binds to a receptor, angiotensin-converting enzyme 2 (ACE2) (9Li 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), through the receptor-binding site of its receptor-binding domain (RBD) (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 10Wong S.K. Li W. Moore M.J. Choe H. Farzan M. J. Biol. Chem. 2004; 279: 3197-3201Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 11Babcock G.J. Esshaki D.J. Thomas Jr., W.D. Ambrosino D.M. J. Virol. 2004; 78: 4552-4560Crossref PubMed Scopus (202) Google Scholar). The RBD is an attractive target for neutralizing antibodies that could prevent SCV entry by blocking the attachment of ACE2 (12He Y. Zhou Y. Liu S. Kou Z. Li W. Farzan M. Jiang S. Biochem. Biophys. Res. Commun. 2004; 324: 773-781Crossref PubMed Scopus (307) Google Scholar, 13Zhang M.Y. Choudhry V. Xiao X. Dimitrov D.S. Curr. Opin. Mol. Ther. 2005; 7: 151-156PubMed Google Scholar, 14Jiang S. He Y. Liu S. Emerg. Infect. Dis. 2005; 11: 1016-1020Crossref PubMed Scopus (174) Google Scholar, 15He Y. Lu H. Siddiqui P. Zhou Y. Jiang S. J. Immunol. 2005; 174: 4908-4915Crossref PubMed Scopus (204) Google Scholar, 16He Y. Zhu Q. Liu S. Zhou Y. Yang B. Li J. Jiang S. Virology. 2005; 334: 74-82Crossref PubMed Scopus (90) Google Scholar, 17Chen Z. Zhang L. Qin C. Ba L. Yi C.E. Zhang F. Wei Q. He T. Yu W. Yu J. Gao H. Tu X. Gettie A. Farzan M. Yuen K.Y. Ho D.D. J. Virol. 2005; 79: 2678-2688Crossref PubMed Scopus (160) Google Scholar, 18Yi C.E. Ba L. Zhang L. Ho D.D. Chen Z. J. Virol. 2005; 79: 11638-11646Crossref PubMed Scopus (50) Google Scholar). To understand the structural mechanisms underlying SCV immunogenicity and neutralization and help in the design of vaccines capable of eliciting predetermined highly effective neutralizing antibodies, we used a retrovaccinology (19Burton D.R. Nat. Rev. Immunol. 2002; 2: 706-713Crossref PubMed Scopus (504) Google Scholar) approach based on the combination of phage display and x-ray crystallography. The SCV is a member of the genus Coronavirus, which belongs to the Coronaviridae family of the order Nidovirales, which also includes families Arteriviridae and Roniviridae. Not only SCV but also other nidoviruses can infect humans and animals, resulting in a variety of severe diseases. The infection is initiated by the attachment of virus envelope glycoproteins (Envs) to receptors, which can be blocked by nAbs. The structural mechanisms of receptor recognition and neutralization by antibodies against any nidovirus were not previously known. Recently, the crystal structure of the SCV S RBD in complex with ACE2 was reported at 2.9-Ä resolution (20Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar). However, the structures of a receptor-free RBD and its complexes with nAbs are not known. Thus, fundamental questions related to the mechanism of SCV (and any other nidovirus) entry and neutralization, such as conformational changes induced by the binding of either the receptor or the nAbs, remain unanswered. To date, only a few structures of viral Envs complexed with nAbs are available, including Envs from influenza, picornaviruses, HIV, and West Nile virus (5Dimitrov D.S. Nat. Rev. Microbiol. 2004; 2: 109-122Crossref PubMed Scopus (393) Google Scholar, 21Nybakken G.E. Oliphant T. Johnson S. Burke S. Diamond M.S. Fremont D.H. Nature. 2005; 437: 764-769Crossref PubMed Scopus (298) Google Scholar); these structures have played an essential role in elucidating the mechanisms of viral neutralization. Here, we describe the identification of a potent cross-reactive SCV-neutralizing human monoclonal antibody, m396, and report the crystal structure of the antibody-antigen complex (Fab m396-SCV RBD) at 2.3-Ä resolution (Supplemental Table S1). The structure reveals a major neutralization determinant and its relationship with receptor recognition, providing structural insights into the mechanism of SCV entry and neutralization. Expression and Purification of the RBD—A fragment containing residues 317–518 from the S glycoprotein was cloned into pSecTag2B (Invitrogen) using BamHI and EcoRI restriction sites as previously described (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 22Chakraborti S. Prabakaran P. Xiao X. Dimitrov D.S. Virol. J. 2005; 2: 73Crossref PubMed Scopus (73) Google Scholar). The insert was further cloned into pAcGP67-A using the forward primer 5′-ACT GTC TAG ATG GTA CCG AGC TCG GAT CC-3′ (XbaI) and the reverse primer 5′-CAG TAG ATC TCG AGG CTG ATC AGC G-3′ (BglII). The pAcGP67-S was co-transfected with BaculoGold linearized baculovirus DNA into SF9 cells. High titer recombinant baculovirus stock was prepared by multiple amplifications. The protein was expressed in SF9 cells, cultured in serum-free HyQ-SFX-insect medium (HyClone), and purified from conditioned medium with a HiTrap nickel-chelating column. The eluted monomeric protein was concentrated, further purified with a Superdex 75 10/300GL column equilibrated with phosphate-buffered saline plus 0.2 m NaCl, and concentrated to 5–10 mg/ml in phosphate-buffered saline plus 0.2 m NaCl. Selection, Expression, and Purification of the High Affinity RBD-specific Fab m396 and Its Conversion to IgG1—A naïve human Fab phage display library (a total of ∼1010 members) was constructed from peripheral blood B cells of 10 healthy donors 5Z. Y. Zhu and D. S. Dimitrov, manuscript in preparation. and used for selection of Fabs against purified, soluble, monomeric RBD, conjugated to magnetic beads (Dynabeads M-270 Epoxy, Dynal Inc., New Hyde Park, NY) following a previously described procedure (23Zhu Z. Dimitrov A.S. Bossart K.N. Crameri G. Bishop K.A. Choudhry V. Mungall B.A. Feng Y.R. Choudhary A. Zhang M.Y. Feng Y. Wang L.F. Xiao X. Eaton B.T. Broder C.C. Dimitrov D.S. J. Virol. 2006; 80: 891-899Crossref PubMed Scopus (127) Google Scholar). Briefly, amplified libraries of 1012 phage-displayed Fabs were incubated with 5, 3, and 1 μg of RBD in a 500-μl volume for 2 h at room temperature during the first, second, an third rounds of biopanning, respectively. After the third round of biopanning, 95 clones were randomly picked from the infected TG1 cells, and phage enzyme-linked immunosorbent assay was used to identify clones of phage displaying Fabs with high binding affinity. Eight clones that bound to the RBD with A450 > 1.0 were selected for further characterization. The VH and VL domains (VH and VL denote the variable domains of heavy and light chains, respectively) of these clones were sequenced. The sequences were identical for all selected clones, and the selected Fab was designated as m396. The Fab used for crystallization was purified with a HiTrap nickel-chelating column followed by a Superdex 75 10/300GL column, using phosphate-buffered saline buffer containing 0.2 m NaCl, and concentrated to 10–20 mg/ml. For its conversion to IgG1, the Fab heavy and light chains were amplified and re-cloned in the pDR12 vector (provided by D. Burton, Scripps Research Institute, La Jolla, CA) with the Fc gene fragment replaced with cDNA sequence instead of genomic DNA. Affinity Determination by Surface Plasmon Resonance—Interactions between m396 and SCV RBD were analyzed by surface plasmon resonance technology using a BIAcore 1000 instrument (Amersham Biosciences). The SCV RBD was covalently immobilized onto a sensor chip (CM5) using carbodiimide coupling chemistry. A control reference surface was prepared for nonspecific binding and refractive index changes. For analysis of the kinetics of interactions, various concentrations of Fab or IgG m396 were injected at a flow rate of 30 μl/min using running buffer containing 150 mm NaCl, 3 mm EDTA, and 0.005% P-20 (pH 7.4). The association and dissociation phase data were fitted simultaneously to a 1:1 Langmuir global model by using the nonlinear data analysis program BIAevaluation 4.1. All the experiments were performed at 25 °C. Crystallization and Structure Determination—The SCV RBD-Fab m396 complex was formed by mixing individual components in a 1:1 molar ratio and incubating overnight at 4 °C. Crystals were obtained within 2–3 weeks by sitting-drop vapor diffusion technique. The reservoir solution was composed of 15% v/v glycerol, 20% polyethylene glycol 6000, 100 mm MES sodium at pH 6.5; crystals formed only in the drops with a 1:2 ratio for the protein and the reservoir solutions. The crystals of Fab m396 were grown with the sitting-drop vapor diffusion technique within 2 weeks. The reservoir solution was composed of 20% v/v glycerol, 16% v/v ethylene glycol, 20% w/v polyethylene glycol 6000, and 100 mm NaCl in 30 mm Tris-HCl (pH 8.5). Data sets up to 2.3-Ä resolution were collected at cryogenic temperature (100 K) for both the RBD-Fab complex and the unliganded Fab, each from a single crystal, at the Southeast Regional Collaborative Access Team beamline facility 22-ID of the Advanced Photon Source, Argonne National Laboratory. Data processing was carried out with the HKL2000 program suite (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). The structure was solved by molecular replacement with PHASER (25Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1099) Google Scholar), using the SCV RBD from the receptor complex (PDB code 2AJF) and four individual domains of Fab (VH,VL,CH, and CL)(CH and CL refer to the constant domains of heavy and light chains, respectively) from three different antibody structures (PDB codes: 1ZA6 for CH and CL, 1RZG for VH, and 1W72 for VL) as search models. The RBM (residues 430–490) of SCV RBD and most of the CDRs (complementarity-determining regions) of Fab models, which were not included in the search models, were built on the basis of difference electron density. The complex was refined with CNS (26Brunger A.T. Adams P.D. Rice L.M. Structure. 1997; 5: 325-336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) at 2.3-Ä resolution. A total of 298 water molecules, a phosphate ion, and one N-linked glucosamine moiety at Asn-330 were added at the final stage of the refinement. The final R and Rfree values were 19.8 and 26.1, respectively. The unliganded Fab m396 structure was solved by molecular replacement with AMoRe (27Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar), using the constant domains of Fab m396 from the complex structure as the search model. The difference electron density map revealed the location of variable domains. The structure was refined using CNS (26Brunger A.T. Adams P.D. Rice L.M. Structure. 1997; 5: 325-336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and a total of 176 water molecules was added at the final stage of the refinement. The final R and Rfree values were 22.8 and 27.7, respectively. The O program (28Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (504) Google Scholar) was used for model building for both structures. Data collection, processing, and refinement statistics are summarized in Supplemental Table S1. Previously, we identified several S glycoprotein fragments, containing the RBD, which is a major SCV neutralization determinant (12He Y. Zhou Y. Liu S. Kou Z. Li W. Farzan M. Jiang S. Biochem. Biophys. Res. Commun. 2004; 324: 773-781Crossref PubMed Scopus (307) Google Scholar, 13Zhang M.Y. Choudhry V. Xiao X. Dimitrov D.S. Curr. Opin. Mol. Ther. 2005; 7: 151-156PubMed Google Scholar, 14Jiang S. He Y. Liu S. Emerg. Infect. Dis. 2005; 11: 1016-1020Crossref PubMed Scopus (174) Google Scholar, 15He Y. Lu H. Siddiqui P. Zhou Y. Jiang S. J. Immunol. 2005; 174: 4908-4915Crossref PubMed Scopus (204) Google Scholar, 16He Y. Zhu Q. Liu S. Zhou Y. Yang B. Li J. Jiang S. Virology. 2005; 334: 74-82Crossref PubMed Scopus (90) Google Scholar, 17Chen Z. Zhang L. Qin C. Ba L. Yi C.E. Zhang F. Wei Q. He T. Yu W. Yu J. Gao H. Tu X. Gettie A. Farzan M. Yuen K.Y. Ho D.D. J. Virol. 2005; 79: 2678-2688Crossref PubMed Scopus (160) Google Scholar, 18Yi C.E. Ba L. Zhang L. Ho D.D. Chen Z. J. Virol. 2005; 79: 11638-11646Crossref PubMed Scopus (50) Google Scholar), and residues critical for the binding of SCV to its receptor ACE2 (7Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 22Chakraborti S. Prabakaran P. Xiao X. Dimitrov D.S. Virol. J. 2005; 2: 73Crossref PubMed Scopus (73) Google Scholar). One of these fragments, containing residues 317–518, was cloned into a baculovirus vector, expressed in insect cells, and purified. This fragment was used as a selecting antigen for panning of a large (∼1010 different antibodies) human antibody Fab library, which we constructed from the B lymphocytes of 10 healthy volunteers. Recently, this library was also used for the selection of potent nAbs against Hendra and Nipah viruses (23Zhu Z. Dimitrov A.S. Bossart K.N. Crameri G. Bishop K.A. Choudhry V. Mungall B.A. Feng Y.R. Choudhary A. Zhang M.Y. Feng Y. Wang L.F. Xiao X. Eaton B.T. Broder C.C. Dimitrov D.S. J. Virol. 2006; 80: 891-899Crossref PubMed Scopus (127) Google Scholar). The Fab with the strongest binding to the RBD, m396, was converted to full antibody (IgG1), expressed, and purified. We measured the binding rate constants and affinities of the Fab and the IgG1 m396 to SCV RBD in a BIAcore assay (Supplemental Fig. S1). With two independent experiments, we determined Kon = 3.0 (±0.3) and 4 (±3) × 105 m–1 s–1, Koff = 6.1 (±0.6) × 10–3 s–1 and2(±1) × 10–5 s–1, and KD = 20 (±0) nm and 4.6 (±0.9) pm, for the Fab and the IgG1 m369, respectively. The high apparent affinity (avidity) observed for IgG1 m369 is due to the effective multivalency of the surface-associated antigen binding to the bivalent IgG1. Further, we found that the antibody potently inhibited 1) cell fusion and pseudovirus entry mediated by the SCV (Tor2 isolate) S glycoprotein with an IC50 of 0.6 and 0.01 μg/ml, respectively, 2) SCV entry mediated by the S glycoprotein from the GD03T0013 isolate, which is not neutralizable by other known human monoclonal antibodies, including 80R (29Sui 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, 30Sui 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) and S3.1 (31Traggiai E. Becker S. Subbarao K. Kolesnikova L. Uematsu Y. Gismondo M.R. Murphy B.R. Rappuoli R. Lanzavecchia A. Nat. Med. 2004; 10: 871-875Crossref PubMed Scopus (569) Google Scholar, 32Yang Z.Y. Werner H.C. Kong W.P. Leung K. Traggiai E. Lanzavecchia A. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 797-801Crossref PubMed Scopus (203) Google Scholar), and 3) live virus from Urbani and Tor2 isolates with an IC50 of 0.1 and 1 μg/ml, respectively. 6Zhu, Z., Prabakaran, P., Gan, J., Chakraborti, S., He, Y., Choudhry, V., Feng, Y., Xiao, X., Wang, L., Ji, X., Jiang, S., and Dimitrov, D. S., manuscript to be submitted. The structure of the SCV RBD-Fab m396 complex is depicted in Fig. 1. The overall RBD structure in the complex with Fab m396 is similar to that in the complex with ACE2 (20Li F. Li W. Farzan M. Harrison S.C. Science. 2005; 309: 1864-1868Crossref PubMed Scopus (1382) Google Scholar). The root mean square deviation between the common Cα positions in the two RBD structures is 1.3 Ä. However, the new RBD structure reveals a total of 16 amino acid residues localized in two segments (residues 376–381 and 503–512 are shown in brown) that were missing in the RBD·ACE2 complex. The antibody-bound RBD structure also reveals a new disulfide bond between residues 378 and 511 (Fig. 2), which was not observed in the RBD·ACE2 complex. As shown in Fig. 1, the RBD consists of a core, which includes a five-stranded anti-parallel β-sheet (β1–β4 and β7), and a long extended loop, which contains a two-stranded anti-parallel β-sheet (β5–β6) in the middle. The complete RBD structure contains eight cysteines that form four disulfide bridges, three in the core and one in the extended loop.FIGURE 2Comparison of the RBD·Fab and the RBD·ACE2 structures. The newly identified two segments of RBD in the Fab complex are denoted by blue and pink colors. a, sequence and secondary structure assignment of RBD. b, structural alignment between RBD structures based on Cα positions. RBD structures from the Fab and ACE2 complexes are shown in green and cyan, respectively. Blue and pink segments defined in the RBD-Fab structure revealed the fourth disulfide bond within the RBD (between residues Cys-378 and Cys-511), which is shown as a stick model. c and d, stereoviews showing the 2Fo – Fc electron density maps contoured at 1.0 σ level along the segments 375–382 and 501–512.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fab m396 mainly recognizes the 10-residue (482–491) β6–β7 loop that prominently protrudes from the RBD surface (Fig. 1) and contacts four of the CDRs of Fab, H1, H2, H3, and L3. The four CDRs form a shallow cleft on the surface of the antibody-variable regions, providing a deep binding pocket into which the β6–β7 loop fits tightly. The tip of this loop is a type I β-turn (Gly-Ile-Gly-Tyr, residues 488–491) and is deeply buried in the antibody-combining site, which is a feature most commonly observed in antibody·peptide complexes (33Wilson I.A. Stanfield R.L. Curr. Opin. Struct. Biol. 1994; 4: 857-867Crossref PubMed Scopus (445) Google Scholar). The same feature involving the recognition of a similar sequence motif (Gly-Pro-Gly-Arg, residues 312–315) has been recently noted at the tip of the gp120 V3 structure (34Huang C.C. Tang M. Zhang M.Y. Majeed S. Montabana E. Stanfield R.L. Dimitrov D.S. Korber B. Sodroski J. Wilson I.A. Wyatt R. Kwong P.D. Science. 2005; 310: 1025-1028Crossref PubMed Scopus (647) Google Scholar), which is a major neutralizing determinant of HIV. Most residues in the β6–β7 loop interact with Fab m396 at the binding pocket, and particularly, residues Ile-489 and Tyr-491 from the β-turn penetrate into the deep pocket on the surface of the antibody-combining site. Fifteen residues from the RBD and the Fab participate in the formation of the RBD-antibody interface as defined by a contact distance of 3.5 Ä between the two molecules. The shape correlation statistical parameter (Sc) (35Lawrence M.C. Colman P.M. J. Mol. Biol. 1993; 234: 946-950Crossref PubMed Scopus (1106) Google Scholar), a measure of geometric fit between two juxtaposed surfaces (maximum value, 1.0), calculated for the RBD-antibody interface is 0.66, indicating a high degree of shape complementarity. A total surface area of 1760 Ä2 is buried at the interface of the complex with nearly equal contributions from the two molecules (870 Ä2 from the RBD and 890 Ä2 from the antibody) as determined with a 1.4-Ä probe. The antibody-binding β6–β7 loop alone accounts for 63% of the RBD-antibody interface, which indicates the dominant role of the loop residues in the binding of the two molecules. The heavy chain CDRs contributes 66% of the total surface of the antibody-combining site. The size of the binding interface is close to the average of other antigen·antibody complexes (36Davies D.R. Cohen G.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7-12Crossref PubMed Scopus (484) Google Scholar). The intermolecular interactions include van der Waals contacts and direct or water-mediated hydrogen bonds (Supplemental Table S2). The details of the buried surface area at the interface between the antibody and the SCV RBD are given in Supplemental Table S3. The key interactions in the RBD·antibody complex are mostly between the β6–β7 loop of the RBD and the four CDRs (H1, H2, H3, and L3) of Fab m396. These interactions are clearly defined (Fig. 3). H1 makes contacts with hydrophobic residues Tyr-484, Thr-486, and Thr-487; particularly Thr-33 of H1 is in direct contact with the amide nitrogen of Gly-488 in RBD (Fig. 3a). A similar interaction is found in the RBD·ACE2 complex where the amide of Gly-488 is engaged in a hydrogen bond with the carbonyl oxygen of Lys-35 in ACE2. Compared with other CDRs, H2 plays a dominant role in RBD binding; the most conspicuous feature is the burial of Tyr-491 of RBD (122 Ä2) in the shallow cleft rendered by H2, where the amino group of Asn-58 contacts the phenolic oxygen atom of Tyr-491 and the side chain of Thr-52 forms an aromatic on-face hydrogen bond with the π -cloud of Tyr-491 (Fig. 3b). Val-97 is the only residue from H3 that interacts with RBD (Fig. 3c); however, it buries the largest surface area per residue (108 Ä2) among all CDR residues that interact with RBD. The carbonyl oxygen atom of Val-97 forms a strong hydrogen bond with the amino nitrogen of the RBD residue Gln-492 with a distance of 2.7 Ä. Such hydrogen bonds between main-chain and side-chain atoms play an important role in determining the relative orientation of the RBD and the antibody in the complex, and contribute to the specificity of the interactions. The Sc parameter calculated for the heavy chain-RBD interaction has a high value of 0.74, which suggests a highly correlated interfacial geometry for the heavy chain-RBD recognition. L3-RBD interaction involves water molecules and additional side chains, including Arg-395, of RBD (Fig. 3d, Supplemental Table S2). Residue Trp-91 of L3 stacks with aromatic residue Ile-489, a major hot spot in the RBD; each of the two residues buries a surface area of ∼100 Ä2 at the interface. The minor binding sites on the RBD include two residues in β2 (Thr-363 and Lys-365), the 310 helix followed by β3 (Lys-390, Gly-391, Asp-392, and Arg-395), and residues Arg-426 and Tyr-436. Apart from the minor contributions of these residues to antibody binding, most of them have significant roles in stabilizing the conformation of the β6–β7 loop. For example, the amide hydrogen atoms of Gly-391 and Asp-392 hydrogen bond to the backbone carbonyls of Gln-492 and Gly-490, respectively; the guanidinium group of Arg-426 forms a hydrogen bond with the carbonyl of Thr-485; and the phenolic oxygen atoms from side chains of Tyr-436 and Tyr-484 are hydrogen-bonded. All these hydrogen bonds help stabilizing the β6–β7 loop conformation in the RBD. The RBD-antibody interface has two major characteristic features: the high level of complementarity between the interacting surfaces and the anchoring of the putative major hotspot RBD residue Tyr-491
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