2021. these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design. One Sentence Summary: X-ray structures of synthetic nanobodies complexed with the receptor-binding domain name of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in acknowledgement of unique epitopic sites. SARS-CoV-2, a -coronavirus, is usually remarkable for its high infectivity, quick worldwide dissemination, and development of highly infectious new variants (1C4). The computer virus exploits its trimeric S glycoprotein to adsorb to the host cell-surface receptor, angiotensin transforming enzyme (ACE) ACE2 (5) resulting in proteolytic processing and conformational changes required for membrane fusion and cell access (6). 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 (7, 8). Exploring the detailed structures of anti-viral antibodies can provide critical understanding of the means to attenuate viral adsorption and access, preventing or retarding ongoing contamination and communal spread. An evolving database of X-ray and cryo-EM structures of the SARS-CoV-2 S and its interactions with ACE2 or numerous antibodies contributes to the design of effective antibodies or immunogens (9). Recent studies indicate the value of single domain name antibodies derived from camelids (nanobodies) (10) or camelid-inspired synthetic libraries (sybodies) (11), and the value of generating multivalent constructs (12) for effective treatment (11). Many properties of nanobodies make them well suited for structural studies and drug development (13). Here, we take advantage of available sequences of three SARS-CoV-2 RBD-directed sybodies – Sb16, Sb45, and Sb68 (previously designated Sb#16, Sb#45, and Sb#68 (14)). We describe binding studies and X-ray structures of complexes of these with the RBD, and also the structure of Sb16 unliganded. The sybodies had been shown to be effective inhibitors of the ACE2CRBD conversation (14), and neutralizers of viral infectivity (14). These sybodies (observe Supplementary Materials and Methods) behaved as monomers by size exclusion chromatography (SEC) (15) (Physique S1), and we confirmed their activity in binding to the re-engineered RBD and S using surface plasmon resonance (SPR) (Physique S2). All three sybodies bind to surface immobilized RBD with insect cells, which produce biantennary N-glycans terminating with N-acetylglucosamine residues (23, 24). Electron density was observed only for the proximal N-glycans at residues N322 and N546, but larger, complex, non-sialylated, biantennary carbohydrates have been detected in glycoproteomic analysis of ACE2 in mammalian cells (25). These are highly flexible carbohydrates adding greater than 1500 Da at each position, so are larger than the single carbohydrate residues visualized in the crystal structure. Additionally, molecular dynamics simulations of RBDCACE2 implicated the direct conversation of carbohydrate with the RBD (26). Thus, the ability of Sb68 to impinge on ACE2 conversation with RBD likely entails the steric clash of the N322- and N546-linked glycans. We also obtained a 2.1 ? structure of free Sb16 (Physique S5). Amazingly, the CDR2 of Sb16 shows Y54 in starkly different positions in the unliganded structure as compared to the complex: Rabbit polyclonal to ACVR2B the C carbon is usually displaced by 6.0 ?, while the O oxygen of Y54 is usually 15.2 ? distant, indicative of dynamic flexibility. To gain additional insight into the structural effects of the interactions of each of these sybodies with a trimeric S protein, we superposed each of the individual sybodyCRBD complexes on each of several cryo-EM-determined models of S, including examples of different combinations of RBD orientation: three-down (6XEY (27)), one-up, two-down (6Z43 (28)), two-up, one-down (7A29 (29)), and three-up Nomilin (7JVC (30)) (observe Physique 4). Both Sb16 and Sb45 may dock on each of the three RBDs in the trimeric S in any of the four configurations, without any apparent clash (Physique 4A, ?,4B).4B). However, Nomilin Sb68 could not be superposed without clashes to any RBD of the three-down or to the one-up two down position. The only permissible superpositions were to two in the two-up, one-down (Physique 4C); and to all three in the three-up position (Physique 4C). For paired sybodies, either Sb16 and Sb68 or Sb45 and Sb68, superposition was possible without clashes, with two or more RBDs in the up conformation (Physique 4D and ?and4E).4E). Walter et al (14) suggested that a covalent bispecific Sb15CSb68 Nomilin reagent could bind S in both the two-up and three-up configurations, based on cryo-EM maps of complexes of S with Sb15 and Sb68, with local resolution in the range of 6C7 ?. It appears Nomilin that Sb16 binds to Nomilin S in an orientation much like, but in detail unique from that of Sb15. This analysis demonstrates an advantage of the small size of sybodies or nanobodies in accessing epitopic regions of S. Open in a separate windows Fig. 4. Superposition of complexes on spike models.
