SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBMtargeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a K D of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies.

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Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the crystal structures of S. aureus PgsA ( Sa PgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria.

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