Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction
2021; Elsevier BV; Volume: 297; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2021.101202
ISSN1083-351X
AutoresJaveed Ahmad, Jiansheng Jiang, Lisa F. Boyd, Allison Zeher, Rick Huang, Di Xia, Kannan Natarajan, David H. Margulies,
Tópico(s)Complement system in diseases
ResumoCombating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures. Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is remarkable for its high infectivity, rapid worldwide dissemination, and evolution of highly infectious new variants (1Conti P. Caraffa A. Gallenga C.E. Kritas S.K. Frydas I. Younes A. Di Emidio P. Tete G. Pregliasco F. Ronconi G. The British variant of the new coronavirus-19 (Sars-Cov-2) should not create a vaccine problem.J. Biol. Regul. Homeost Agents. 2021; 35: 1-4PubMed Google Scholar, 2Kirby T. New variant of SARS-CoV-2 in UK causes surge of COVID-19.Lancet Respir. Med. 2021; 9: e20-e21Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 3Tang J.W. Tambyah P.A. Hui D.S.C. Emergence of a novel coronavirus causing respiratory illness from Wuhan, China.J. Infect. 2020; 80: 350-371Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 4Wibmer C.K. Ayres F. Hermanus T. Madzivhandila M. Kgagudi P. Oosthuysen B. Lambson B.E. de Oliveira T. Vermeulen M. van der Berg K. Rossouw T. Boswell M. Ueckermann V. Meiring S. von Gottberg A. et al.SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma.Nat. Med. 2021; 27: 622-625Crossref PubMed Scopus (671) Google Scholar). The virus exploits its trimeric spike (S) glycoprotein to adsorb to the host cell-surface receptor, angiotensin-converting enzyme 2 (ACE2) (5Wang Q. Zhang Y. Wu L. Niu S. Song C. Zhang Z. Lu G. Qiao C. Hu Y. Yuen K.Y. Wang Q. Zhou H. Yan J. Qi J. Structural and functional basis of SARS-CoV-2 entry by using human ACE2.Cell. 2020; 181: 894-904.e9Abstract Full Text Full Text PDF PubMed Scopus (1886) Google Scholar), resulting in proteolytic processing and conformational changes required for membrane fusion and cell entry (6Shang J. Wan Y. Luo C. Ye G. Geng Q. Auerbach A. Li F. Cell entry mechanisms of SARS-CoV-2.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 11727-11734Crossref PubMed Scopus (2010) Google Scholar). Understanding the fundamental molecular and cell biology and chemistry of the viral life cycle and the nature of the host immune response offers rational avenues for developing diagnostics, therapeutics, and vaccines (7Baden L.R. El Sahly H.M. Essink B. Kotloff K. Frey S. Novak R. Diemert D. Spector S.A. Rouphael N. Creech C.B. McGettigan J. Khetan S. Segall N. Solis J. Brosz A. et al.Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.N. Engl. J. Med. 2021; 384: 403-416Crossref PubMed Scopus (5763) Google Scholar, 8Cao Y. Su B. Guo X. Sun W. Deng Y. Bao L. Zhu Q. Zhang X. Zheng Y. Geng C. Chai X. He R. Li X. Lv Q. Zhu H. et al.Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients' B cells.Cell. 2020; 182: 73-84.e16Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). Emerging viral variants that exhibit increased infectivity and virulence emphasize the need for continued improvement in immunization and therapeutic approaches. Specifically, B.1.1.7 (United Kingdom), B.1.351 (South Africa), P.1 (Brazil), and other strains demand careful attention (9Cerutti G. Rapp M. Guo Y. Bahna F. Bimela J. Reddem E.R. Yu J. Wang P. Liu L. Huang Y. Ho D.D. Kwong P.D. Sheng Z. Shapiro L. Structural basis for accommodation of emerging B.1.351 and B.1.1.7 variants by two potent SARS-CoV-2 neutralizing antibodies.Structure. 2021; 29: 655-663.e4Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 10Edara V.V. Norwood C. Floyd K. Lai L. Davis-Gardner M.E. Hudson W.H. Mantus G. Nyhoff L.E. Adelman M.W. Fineman R. Patel S. Byram R. Gomes D.N. Michael G. Abdullahi H. et al.Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.02.20.432046Crossref Google Scholar, 11Planas D. Bruel T. Grzelak L. Guivel-Benhassine F. Staropoli I. Porrot F. Planchais C. Buchrieser J. Rajah M.M. Bishop E. Albert M. Donati F. Prot M. Behillil S. Enouf V. et al.Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies.Nat. Med. 2021; 27: 917-924Crossref PubMed Scopus (428) Google Scholar, 12Ramanathan M. Ferguson I.D. Miao W. Khavari P.A. SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity.Lancet Infect. Dis. 2021; 21: 1070Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Singh J. Samal J. Kumar V. Sharma J. Agrawal U. Ehtesham N.Z. Sundar D. Rahman S.A. Hira S. Hasnain S.E. Structure-function analyses of new SARS-CoV-2 variants B.1.1.7, B.1.351 and B.1.1.28.1: Clinical, diagnostic, therapeutic and public health implications.Viruses. 2021; 13: 439Crossref PubMed Scopus (81) Google Scholar, 14Wang P. Nair M.S. Liu L. Iketani S. Luo Y. Guo Y. Wang M. Yu J. Zhang B. Kwong P.D. Graham B.S. Mascola J.R. Chang J.Y. Yin M.T. Sobieszczyk M. et al.Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7.Nature. 2021; 593: 130-135Crossref PubMed Scopus (1331) Google Scholar). Exploring the detailed structures of antiviral antibodies can provide critical understanding of the means to attenuate viral adsorption and entry and prevent or retard ongoing infection and communal spread. An evolving database of X-ray and cryo-EM structures of the SARS-CoV-2 S and receptor-binding domain (RBD) and their interactions with ACE2 or various antibodies contributes to the design of effective antibodies or immunogens (15Xu C. Wang Y. Liu C. Zhang C. Han W. Hong X. Wang Y. Hong Q. Wang S. Zhao Q. Wang Y. Yang Y. Chen K. Zheng W. Kong L. et al.Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM.Sci. Adv. 2021; 7eabe5575Crossref PubMed Scopus (208) Google Scholar). Recent studies indicate the value of single-domain antibodies derived from camelids (nanobodies) (16Hanke L. Vidakovics Perez L. Sheward D.J. Das H. Schulte T. Moliner-Morro A. Corcoran M. Achour A. Karlsson Hedestam G.B. Hallberg B.M. Murrell B. McInerney G.M. An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction.Nat. Commun. 2020; 11: 4420Crossref PubMed Scopus (181) Google Scholar) or camelid-inspired synthetic libraries (sybodies) (17Schoof M. Faust B. Saunders R.A. Sangwan S. Rezelj V. Hoppe N. Boone M. Billesbolle C.B. Puchades C. Azumaya C.M. Kratochvil H.T. Zimanyi M. Deshpande I. Liang J. Dickinson S. et al.An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive spike.Science. 2020; 370: 1473-1479Crossref PubMed Scopus (232) Google Scholar, 18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar) and the potential effectiveness of multivalent constructs (19Xiang Y. Nambulli S. Xiao Z. Liu H. Sang Z. Duprex W.P. Schneidman-Duhovny D. Zhang C. Shi Y. Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2.Science. 2020; 370: 1479-1484Crossref PubMed Scopus (210) Google Scholar). Many properties of nanobodies make them well suited for structural studies and drug development (20Ingram J.R. Schmidt F.I. Ploegh H.L. Exploiting nanobodies' singular traits.Annu. Rev. Immunol. 2018; 36: 695-715Crossref PubMed Scopus (135) Google Scholar). Here, we take advantage of available sequences of five SARS-CoV-2 RBD–directed sybodies: Sb14, Sb15, Sb16, Sb45, and Sb68 (previously designated Sb#14, Sb#15, Sb#16, Sb#45, and Sb#68, respectively (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar)). These sybodies effectively inhibit the ACE2–RBD interaction and neutralize viral infectivity (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar), and a bispecific construct, consisting of Sb15 linked to Sb68, blocked ACE2 binding and neutralized both pseudotyped and infectious SARS-CoV-2 viruses (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar). Here, we describe binding studies and X-ray structures of binary complexes of Sb16–RBD and Sb45–RBD, ternary complexes of Sb14–RBD–Sb68 and Sb45–RBD–Sb68, and unliganded Sb16. In addition, we report cryo-EM structures of Sb45 complexed with trimeric S and evaluate sybody interactions with several mutant RBDs, representative of newly evolving variants. Sybodies were expressed in Escherichia coli and purified via metal-affinity chromatography to high purity. These sybodies behaved as monomers by size-exclusion chromatography (SEC) (21Zimmermann I. Egloff P. Hutter C.A.J. Kuhn B.T. Brauer P. Newstead S. Dawson R.J.P. Geertsma E.R. Seeger M.A. Generation of synthetic nanobodies against delicate proteins.Nat. Protoc. 2020; 15: 1707-1741Crossref PubMed Scopus (72) Google Scholar) (Fig. S1), and we confirmed their activity in binding to the bacteria-expressed RBD as visualized by SEC (Fig. S1). As determined by surface plasmon resonance (SPR), all five sybodies bind to the immobilized RBD with KD values of 6.8 to 62.7 nM, consistent with previous determinations using RBD-YFP or RBD-Fc molecules by related techniques (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar) (Fig. 1). To gain insight into the precise topology of the interaction of four of these sybodies with the RBD, we determined crystal structures of their complexes: Sb16–RBD and Sb45–RBD, the ternary Sb45–RBD–Sb68 and Sb14–RBD–Sb68, and Sb16 alone. These crystals diffracted X-rays to resolutions from 1.7 to 2.6 Å (Table 1). After molecular replacement, model building, and crystallographic refinement (see Experimental procedures), we obtained structural models that satisfied standard criteria for fitting and geometry (Table 1). Illustrations of the quality of the final models as compared with the electron density maps are shown in Fig. S2.Table 1X-ray data collection and refinement statisticsData collection and refinementSb16–RBDSb45–RBDSb14–RBD–Sb68Sb45–RBD–Sb68Sb16PDB ID7KGK7KGJ7MFU7KLW7MFVData collection Space groupP6522P3221P21C2221P6322 Cell dimensionsa, b, c (Å)aValues in parentheses are for the highest resolution shell.65.64, 65.64, 344.6962.55, 62.55, 168.8266.82, 83.05, 92.8374.50, 102.40, 138.9768.92, 68.92, 107.17α, β, γ (°)90.0, 90.0, 120.090.0, 90.0, 120.090.0, 106.71, 90.090.0, 90.0, 90.090.0, 90.0, 120.0 Resolution (Å)57.34–2.60 (2.69–2.60)45.59–2.30 (2.38–2.30)42.17–1.70 (1.76–1.70)44.12–2.60 (2.69–2.60)34.46–1.90 (1.97–1.90) Rsym or Rmerge0.080 (0.455)0.101 (0.849)0.086 (0.765)0.095 (0.739)0.074 (1.54) I/σ(I)18.0 (3.3)14.9 (3.4)8.9 (1.7)13.1 (2.1)15.2 (0.7) Completeness (%)98.8 (99.1)99.3 (98.3)98.4 (93.8)98.8 (98.7)94.5 (85.0) Redundancy10.3 (10.9)7.9 (8.2)3.1 (3.1)7.2 (7.4)12.4 (12.6) Rpim0.024 (0.134)0.038 (0.293)0.057 (0.510)0.038 (0.287)0.022 (1.05) CC1/20.999 (0.987)0.997 (0.919)0.995 (0.640)0.998 (0.895)0.999 (0.526) Estimated twin fraction0.0 (none)0.06 (−h, −k, l)0.0 (none)0.0 (none)0.0 (none)Refinement Resolution (Å)56.09–2.60 (2.69–2.60)45.59–2.30 (2.38–2.30)42.27–1.70 (1.76–1.70)36.72–2.60 (2.69–2.60)34.46–1.90 (1.97–1.90) No. of reflections13,219 (1185)17,592 (1687)105,129 (9993)16,508 (1627)11,786 (1025) Rwork/Rfree (%)25.8/27.7 (36.3/44.2)18.6/21.6 (24.1/29.8)18.1/21.5 (27.0/31.6)20.6/25.5 (29.3/34.5)23.7/25.1 (35.4/36.7) No. of atoms2486264177983552987Protein2486250067983456913Water + ligands0141962 + 389670 + 4 B-factor Wilson/Average39.3/59.826.9/32.920.3/26.933.9/31.531.8/44.9Protein59.832.825.731.544.8Water + ligands034.734.5 + 40.029.545.4 + 57.0 RMSDsBond length (Å)0.0020.0050.0040.0030.006Bond angle (°)0.540.740.740.640.96 RamachandranFavored (%)92.997.498.396.394.7Allowed (%)7.12.61.53.75.3Outliers (%)0.00.00.20.00.0 MolProbityClashscore (percentile)5.35 (99th)4.94 (99th)1.8 (99th)5.95 (99th)6.75 (92nd)a Values in parentheses are for the highest resolution shell. Open table in a new tab The structure of the RBD of these complexes (Fig. 2, A and B) revealed little difference between insect-expressed (22Wu Y. Wang F. Shen C. Peng W. Li D. Zhao C. Li Z. Li S. Bi Y. Yang Y. Gong Y. Xiao H. Fan Z. Tan S. Wu G. et al.A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2.Science. 2020; 368: 1274-1278Crossref PubMed Scopus (680) Google Scholar) and our bacteria-expressed and refolded RBDs. Each of the sybodies represents an immunoglobulin variable-type fold (23Natarajan K. Mage M.G. Margulies D.H. Immunoglobulin superfamily.eLS. 2015; https://doi.org/10.1002/9780470015902.a0000926.pub2Crossref Google Scholar, 24Halaby D.M. Poupon A. Mornon J. The immunoglobulin fold family: Sequence analysis and 3D structure comparisons.Protein Eng. 1999; 12: 563-571Crossref PubMed Scopus (196) Google Scholar) consisting of two β-sheets packed as a β-barrel linked by a disulfide bond. The Sb16–RBD complex (Figs. 2A and 3A) illustrates that complementarity-determining region 2 (CDR2) (residues 50–60) and complementarity-determining region 3 (CDR3) (residues 98–106) bestride the saddle-like region of the ACE2-binding surface of the RBD (see sequence alignment in Fig. 2F). Sb16 angulates over the RBD by 83°. However, Sb45 (Figs. 2B and 3B) straddles the RBD saddle in the opposite orientation, at an angle of −36°, and frames the interface with CDR2 (residues 50–59) and CDR3 (residues 97–111). Complementarity-determining region 1 (CDR1) of both sybodies (residues 27–35) lies between the CDR2 and CDR3 loops. Superposition of the two structures, based on the RBD, emphasizes the diametrically opposite orientation of the two (Fig. 2C), revealing that CDR2 of Sb16 and CDR3 of Sb45 recognize the same epitopic regions.Figure 3Interfaces and interactions of sybodies with the RBD. A, Sb16–RBD; (B) Sb45–RBD; (C) Sb14–RBD; and (D) Sb68–RBD. (Individual contacting residues are listed in Table S1). CDR1, CDR2, and CDR3 are painted pink, orange, and red, respectively. Additional non-CDR-contacting residues are colored lime. On the RBD surface, the epitopic residues that contact the sybodies are colored according to the sybody CDR. CDR1, complementarity-determining region 1; CDR2, complementarity-determining region 2; CDR3, complementarity-determining region 3; RBD, receptor-binding domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By exploring the conditions using mixtures of two or three sybodies and the RBD, we obtained crystals and solved the structures of ternary complexes consisting of Sb45–RBD–Sb68 at 2.6 Å resolution (Table 1 and Fig. 2D) and Sb14–RBD–Sb68 at 1.7 Å resolution (Fig. 2E). The refined models revealed that while Sb14 and Sb45 interact with the ACE2 interface of the RBD, Sb68 binds the RBD at a distinct site (Fig. 2, D and E). In the ternary complex, Sb45 binds in an identical orientation to that observed in the binary Sb45–RBD structure (RMSD of superposition, 0.491 Å for 1981 atoms), but Sb68 addresses a completely different face of the RBD, similar to that bound by Fab of CR3022 on the RBD of SARS-CoV-2 (25Huo J. Zhao Y. Ren J. Zhou D. Duyvesteyn H.M.E. Ginn H.M. Carrique L. Malinauskas T. Ruza R.R. Shah P.N.M. Tan T.K. Rijal P. Coombes N. Bewley K.R. Tree J.A. et al.Neutralization of SARS-CoV-2 by destruction of the prefusion spike.Cell Host Microbe. 2020; 28: 445-454.e6Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and by VHH72 on the RBD of SARS-CoV-1 (26Wrapp D. De Vlieger D. Corbett K.S. Torres G.M. Wang N. Van Breedam W. Roose K. van Schie L. Team V.-C.C.-R. Hoffmann M. Pohlmann S. Graham B.S. Callewaert N. Schepens B. Saelens X. et al.Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies.Cell. 2020; 181: 1004-1015.e15Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Of particular interest, whereas Sb45 CDR2 and CDR3 span the RBD saddle as noted above, the distinct contacts of Sb68 to the RBD are through the longer CDR3, with only minor contributions from CDR1 and CDR2. Walter et al. (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar) visualized similar distinct interactions in cryo-EM maps of two sybodies (Sb15 and Sb68) bound to S protein with local resolution of 6 to 7 Å. Similarly, Sb14, which interacts via distinct sybody residues with the RBD at the ACE2 site (see description below), still permits Sb68 to bind to its epitope as seen in the Sb45–RBD–Sb68 structure (Fig. 2D). Scrutiny of the different interfaces provides insights into the distinct ways each sybody exploits its unique CDR residues for interaction with epitopic residues of the RBD (Fig. 3). (Compilation of the contacting residues for each of the four sybodies to the RBD is provided in Table S1). Both Sb16 and Sb45 use longer CDR2 and CDR3 to straddle the RBD, positioning CDR1 residues over the central crest of the saddle (Figs. 2, A–C and 3, A and B and Table S1). In addition, several non-CDR residues (Y37, E44, and W47 for Sb16), derived from framework 2 (27Wu T.T. Kabat E.A. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity.J. Exp. Med. 1970; 132: 211-250Crossref PubMed Scopus (947) Google Scholar), provide additional contacts to the RBD (see Table S1). In contrast with Sb16 and Sb45, Sb14, despite interacting with a large surface area of the RBD, uses both CDR2 and CDR3 on the same side and exploits many non-CDR residues, particularly sheets of β-strand as its binding surface (Fig. 3C and Table S1). The interface of Sb68 with the RBD (Fig. 3D) is quite different, predominantly exploiting nine CDR3, four CDR2, and one CDR1 residues at the interface (see Table S1). To evaluate the structural basis for the ability of these four sybodies to block the interaction of the RBD with ACE2, we superposed each of the three sybody–RBD structures onto the ACE2–RBD structure and examined the steric clashes (Fig. 4A). Sb16 and Sb45 directly impinge on the ACE2 binding site, offering a structural rationale for their viral neutralization capacity (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar). Sb68, which also blocks viral infectivity, binds to the RBD at a site that appears to be noncompetitive for ACE2 binding. The carbohydrate at ACE2 residues N322 and N546 provides an explanation (Fig. 4A). To compare the epitopic areas captured by these sybodies, we evaluated the buried surface area (BSA) interfaces between the RBD and ACE2 or the sybodies. The BSA at the ACE2–RBD, Sb14–RBD, Sb16–RBD, Sb45–RBD, and Sb68–RBD interfaces is 844 Å2, 1040 Å2, 1003 Å2, 976 Å2, and 640 Å2, respectively (Fig. 3, A–E). Sb16 and Sb45 capture more surface area than ACE2 or other published nanobody or sybody–RBD complexes (see Table S2). The interface with Sb68 is the smallest (640 Å2) (Fig. 3D). The total BSA captured by Sb45 and Sb68 in the ternary complex is 1650 (1010 plus 640) Å2 (Table S2) and is consistent with the view that a linked bispecific sybody, as described by Walter et al. (18Walter J.D. Hutter C.A.J. Garaeva A.A. Scherer M. Zimmermann I. Wyss M. Rheinberger J. Ruedin Y. Earp J. Egloff P. Sorgenfrei M. Hürlimann L.M. Gonda I. Meier G. Remm S. et al.Highly potent bispecific sybodies neutralize SARS-CoV-2.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.11.10.376822Crossref Scopus (0) Google Scholar), would exert strong avidity effects. Table S2 summarizes these BSA values and those of other nanobody–RBD interactions. Although Sb68 reveals the smallest BSA with the RBD and binds at a distinct site, it still blocks ACE2 binding. A reasonable explanation for the ability of Sb68 to block the ACE2–RBD interaction arises on inspection of the sites where Sb68, bound to the RBD, might clash with ACE2. Scrutiny of a superposition of Sb68–RBD with ACE2–RBD reveals several areas of steric interference. Sb68 loops 40 to 44 clash with amino acid side chains of ACE2 (residues 318–320 and 548–552), loops 61 to 64 with ACE2 N322 carbohydrate, and loops 87 to 89 (a 310 helix) with ACE2 N546 carbohydrate as well as residues 313 and 316 to 218 (Fig. 4A). ACE2 used in the crystallographic visualization of ACE2–RBD (28Lan J. Ge J. Yu J. Shan S. Zhou H. Fan S. Zhang Q. Shi X. Wang Q. Zhang L. Wang X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.Nature. 2020; 581: 215-220Crossref PubMed Scopus (3629) Google Scholar) was expressed in Trichoplusia ni insect cells, which produce biantennary N-glycans terminating with N-acetylglucosamine residues (29Rudd P.M. Downing A.K. Cadene M. Harvey D.J. Wormald M.R. Weir I. Dwek R.A. Rifkin D.B. Gleizes P.E. Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells.Biochemistry. 2000; 39: 1596-1603Crossref PubMed Scopus (35) Google Scholar, 30Hsu T.A. Takahashi N. Tsukamoto Y. Kato K. Shimada I. Masuda K. Whiteley E.M. Fan J.Q. Lee Y.C. Betenbaugh M.J. Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells.J. Biol. Chem. 1997; 272: 9062-9070Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Electron density was observed only for the proximal N-glycans at residues N322 and N546, but larger, complex, nonsialylated, and biantennary carbohydrates have been detected in glycoproteomic analysis of ACE2 in mammalian cells (31Shajahan A. Archer-Hartmann S. Supekar N.T. Gleinich A.S. Heiss C. Azadi P. Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.Glycobiology. 2021; 31: 410-424Crossref PubMed Scopus (72) Google Scholar). These carbohydrates are highly flexible, adding greater than 1500 Da at each position and are larger than the single carbohydrate residues visualized in the crystal structure. In addition, molecular dynamics simulations of ACE2–RBD implicated the direct interaction of carbohydrate with the RBD (32Zhao P. Praissman J.L. Grant O.C. Cai Y. Xiao T. Rosenbalm K.E. Aoki K. Kellman B.P. Bridger R. Barouch D.H. Brindley M.A. Lewis N.E. Tiemeyer M. Chen B. Woods R.J. et al.Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor.Cell Host Microbe. 2020; 28: 586-601.e6Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Thus, the ability of Sb68 to impinge on ACE2 interaction with the RBD likely involves the steric clash of the N322- and N546-linked glycans. We also obtained a 1.9 Å structure of free Sb16 (Fig. S3). Remarkably, CDR2 of Sb16 shows Y54 in starkly different positions in the unliganded structure as compared with the complex: the Cα carbon is displaced by 6.0 Å, while the Oη oxygen of Y54 is 15.2 Å distant, indicative of dynamic flexibility. To gain further insight into the interaction of Sb45 with the full S protein, we prepared complexes of Sb45 with HexaPro S (S-6P), a stable S variant containing six beneficial proline substitutions (33Hsieh C.L. Goldsmith J.A. Schaub J.M. DiVenere A.M. Kuo H.C. Javanmardi K. Le K.C. Wrapp D. Lee A.G. Liu Y. Chou C.W. Byrne P.O. Hjorth C.K. Johnson N.V. Ludes-Meyers J. et al.Structure-based design of prefusion-stabilized SARS-CoV-2 spikes.Science. 2020; 369: 1501-1505Crossref PubMed Scopus (4) Google Scholar) and acquired cryo-EM images as described in Experimental procedures. All image processing, 2D class, 3D reconstruction, and map refinements were performed with cryoSPARC (34Punjani A. Fleet D.J. 3D variability analysis: Resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM.J. Struct. Biol. 2021; 213: 107702Crossref PubMed Scopus (276) Google Scholar, 35Punjani A. Fleet D.J. 3D flexible refinement: Structure and motion of flexible proteins from cryo-EM.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.04.22.440893Crossref Scopus (0) Google Scholar, 36Punjani A. Rubinstein J.L. Fleet D.J. Brubaker M.A. cryoSPARC: Algorithms for rapid unsupervised cryo-EM structure determination.Nat. Methods. 2017; 14: 290-296Crossref PubMed Scopus (2841) Google Scholar, 37Punjani A. Zhang H. Fleet D.J. Non-uniform refinement: Adaptive regularization improves single-particle cryo-EM reconstruction.Nat. Methods. 2020; 17: 1214-1221Crossref PubMed Scopus (376) Google Scholar), model fitting with Chimera, (38Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera--a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (30045) Google Scholar) and refinement with PHENIX (39Adams P.D. Afonine P.V. Bunkoczi G. Chen V.B. Davis I.W. Echols N. Headd J.J. Hung L.W. Kapral G.J. Grosse-Kunstleve R.W. McCoy A.J. Moriarty N.W. Oeffner R. Read R.J. Richardson D.C. et al.PHENIX: A comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 213-221Crossref PubMed Scopus (17249) Google Scholar). We identified two conformations of S-6P with the RBD in either a 1-up, 2-down (7N0G/EMD-24105) or 2-up, 1-down (7N0H/EMD-24106) position as determined by 3D classification (3D ab initio reconstruction) (Fig. S4). We have built in additional loops of the N-terminal domain (NTD) and glycans based on the models of 6XKL, 7KGJ, and 7B62. We used unsharpened maps for model refinement. The overall correlation coefficients (CCs) (mask/volume/peaks) of models for 7N0G and 7N0H are 0.84/0.84/0.77 and 0.83/0.83/0.77, respectively. The model quality is shown in Table 2. There are three Sb45s binding to
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