key: cord-0757357-u41feo7r
authors: Ahmad, Javeed; Jiang, Jiansheng; Boyd, Lisa F.; Zeher, Allison; Huang, Rick; Xia, Di; Natarajan, Kannan; Margulies, David H.
title: Structures of synthetic nanobody–SARS-CoV-2–RBD complexes reveal distinct sites of interaction and recognition of variants
date: 2021-06-16
journal: Res Sq
DOI: 10.21203/rs.3.rs-625642/v1
sha: 0bc952b270ea1bf9409f68459465af80702b92c7
doc_id: 757357
cord_uid: u41feo7r

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of new variants demands understanding 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 binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68; and Sb16 unliganded. These reveal that Sb14, Sb16, and Sb45 bind the RBD at the ACE2 interface and that the Sb16 interaction is accompanied by a large CDR2 shift. In contrast, Sb68 interacts at the periphery of the interface. We also determined cryo-EM structures of Sb45 bound to spike (S). Superposition of the X-ray structures of sybodies onto the trimeric S protein cryo-EM map indicates some may bind both “up” and “down” configurations, but others may not. Sensitivity of sybody binding to several recently identified RBD mutants is consistent with these structures.

6.8 to 62.7 nM, consistent with previous determinations using RBD-YFP or RBD-Fc molecules 60 by related techniques 18 (Fig.1) . 61 Structure of Sybody-RBD binary and ternary complexes. To gain insight into the precise 62 topology of the interaction of four of these sybodies with the RBD, we determined crystal 63 structures of their complexes: Sb16-RBD, Sb45-RBD; the ternary Sb45-RBD-Sb68 and Sb14-64 RBD-Sb68; and of Sb16 alone. These crystals diffracted X-rays to resolutions from 1.7 to 2.6 Å 65 (Table 1) . After molecular replacement, model building, and crystallographic refinement (see 66 Methods), we obtained structural models that satisfied standard criteria for fitting and geometry 67 ( Table 1) . Illustrations of the quality of the final models as compared with the electron density 68 maps are shown in Extended Data Fig. 2 . 69 The structure of the RBD of these complexes (Fig. 2a, b) revealed little difference between 70 insect-expressed 22 and our bacteria-expressed and refolded RBD. Each of the sybodies has a barrel 71 of two β-sheets stabilized by a single disulfide-linked loop of 75 or 76 amino acids characteristic 72 of an IgV fold 23,24 . The Sb16-RBD complex (Fig. 2a, 3a) illustrates that CDR2 (residues 50-60) 73 and CDR3 (residues 98-106) bestride the saddle-like region of the ACE2-binding surface of the 74 RBD (see sequence alignment in Fig. 2f ). Sb16 angulates over the RBD by 83°. However, Sb45 75 ( Fig. 2b and 3b ) straddles the RBD saddle in the opposite orientation, at an angle of -36°, and 76 frames the interface with CDR2 (residues 50-59) and CDR3 (residues 97-111). CDR1s of both 77 sybodies (residues 27-35) lie between the CDR2 and CDR3 loops. Superposition of the two 78 structures, based on the RBD, emphasizes the diametrically opposite orientation of the two (Fig.   79 2c), revealing that the CDR2 of Sb16 and CDR3 of Sb45 recognize the same epitopic regions. 80 Exploring conditions using mixtures of two or three sybodies and the RBD, we obtained 81 crystals and solved the structures of ternary complexes consisting of Sb45-RBD-Sb68 at 2.6 Å 82 resolution (Table 1 and Fig. 2d ) and Sb14-RBD-Sb68 at 1.7 Å resolution (Fig. 2e) . The refined 83 models revealed that while Sb14 and Sb45 interact with the ACE2 interface of the RBD, Sb68 84 binds the RBD at a distinct site (Fig. 2d, e) . In the ternary complex, Sb45 binds in an identical 85 orientation to that observed in the binary Sb45-RBD structure (RMSD of superposition, 0.491 Å 86 for 1981 atoms), but Sb68 addresses a completely different face of the RBD -similar to that bound Sb14, which interacts via distinct sybody residues with the RBD at the ACE2 site (see description 93 below), still permits Sb68 to bind to its epitope as seen in the Sb45-RBD-Sb68 structure (Fig. 2d ). 94 Scrutiny of the different interfaces provides insights into the distinct ways each sybody 95 exploits its unique CDR residues for interaction with epitopic residues of the RBD (Fig. 3) . 96 (Compilation of the contacting residues for each of the four sybodies to the RBD is provided in 97 Supplementary Table 1) . Both Sb16 and Sb45 use longer CDR2 and CDR3 to straddle the RBD, Table 1) . Also, several non-CDR residues (Y37, E44, and W47 for Sb16), derived 100 from framework 2 27 , provide additional contacts to the RBD (see Supplementary Table 1) . By 101 contrast with Sb16 and Sb45, Sb14, despite interacting with a large surface area of the RBD, uses 102 both CDR2 and CDR3 on the same side and exploits many non-CDR residues, particularly sheets 103 of b-strand as its binding surface ( Fig. 3c and Supplementary Table 1 ). The interface of Sb68 with 104 RBD (Fig. 3d) is quite different, predominantly exploiting nine CDR3, four CDR2, and one CDR1 105 residues at the interface (see Supplementary Table 1) .

Sybodies block ACE2-RBD interaction in discrete ways. To evaluate the structural basis for the 107 ability of these four sybodies to block the interaction of RBD with ACE2, we superposed each of 108 three sybody-RBD structures onto the ACE2-RBD structure and examined the steric clashes ( Fig.   109 4a). Sb16 and Sb45 directly impinge on the ACE2 binding site, offering a structural rationale for 110 their viral neutralization capacity 18 . Sb68, which also blocks viral infectivity, binds to RBD at a 111 site which appears to be noncompetitive for ACE2 binding. The carbohydrate at ACE2 residues 112 N322 and N546 provides an explanation (Fig. 4a ).

To compare the epitopic areas captured by these sybodies, we evaluated the buried surface 114 area (BSA) interfaces between RBD and ACE2 or the sybodies. The BSA at the ACE2-RBD, 115 Sb14-RBD, Sb16-RBD, Sb45-RBD, and Sb68-RBD interfaces are 844 Å 2 , 1,040 Å 2 , 1,003 Å 2 , 116 976 Å 2 , and 640 Å 2 , respectively ( Fig. 3a- (Fig. 4a) . The ACE2 used in the 130 crystallographic visualization of ACE2-RBD 28 was expressed in Trichoplusia ni insect cells, 131 which produce biantennary N-glycans terminating with N-acetylglucosamine residues 29,30 .

Electron density was observed only for the proximal N-glycans at residues N322 and N546, but 133 larger, complex, non-sialylated, biantennary carbohydrates have been detected in glycoproteomic 134 analysis of ACE2 in mammalian cells 31 . These carbohydrates are highly flexible, adding greater 135 than 1500 Da at each position, and are larger than the single carbohydrate residues visualized in 136 the crystal structure. Additionally, molecular dynamics simulations of ACE2-RBD implicated the 137 direct interaction of carbohydrate with the RBD 32 . Thus, the ability of Sb68 to impinge on ACE2 138 interaction with RBD likely involves the steric clash of the N322-and N546-linked glycans. 139 We also obtained a 1.9 Å structure of free Sb16 (Extended Data Fig. 3) . Remarkably, the However, unlike the other class 4 antibodies, Sb68 competes presumably due to its spatial 225 orientation. Overall, our structural studies not only define the Sb14, Sb16, Sb45, and Sb68 epitopes 226 at high resolution, but also reveal that these sybodies capture a rather large epitopic area 227 (Supplementary Table 2 ), suggesting that a judicious choice of several sybodies or nanobodies 228 have the potential to effectively saturate the available RBD surface.

The significance of the ternary structures of Sb45-RBD-Sb68 (7KLW) and Sb14-RBD- Sb68 (Extended Data Fig. 9 ).

Crystallization, data collection, structure determination and crystallographic refinement.

Purified sybodies (Sb14, Sb15, Sb16, Sb45 and Sb68) and RBD were mixed in approximate 1:1 333 molar ratio to a final concentration of 8 mg/ml. The protein mixtures were incubated on ice for 1 334 hour prior to screening. Initial screening for crystals was carried out using the hanging drop vapor with Rwork/Rfree 22.4/25.9. Data collection and structure refinement statistics are provided in Table   366 1. Table 2 . 

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We appreciate the help of Joy (Huaying) Zhao and Peter Schuck, NIBIB, NIH

This is a list of supplementary les associated with this preprint. Click to download.AhmadetalSuppTablesExtData.pdf