A potent and broad-spectrum neutralizing nanobody for SARS-CoV-2 viruses, including all major Omicron strains.

SARS-CoV-2 viruses are highly transmissible and immune evasive. It is critical to develop broad-spectrum prophylactic and therapeutic antibodies for potential future pandemics. Here, we used the phage display method to discover nanobodies (Nbs) for neutralizing SARS-CoV-2 viruses especially Omicron strains. The leading nanobody (Nb), namely, Nb4, with excellent physicochemical properties, can neutralize Delta and Omicron subtypes, including BA.1, BA.1.1 (BA.1 + R346K), BA.2, BA.5, BQ.1, and XBB.1. The crystal structure of Nb4 in complex with the receptor-binding domain (RBD) of BA.1 Spike protein reveals that Nb4 interacts with an epitope on the RBD overlapping with the receptor-binding motif, and thus competes with angiotensin-converting enzyme 2 (ACE2) binding. Nb4 is expected to be effective for neutralizing most recent Omicron variants, since the epitopes are evolutionarily conserved among them. Indeed, trivalent Nb4 interacts with the XBB1.5 Spike protein with low nM affinity and competes for ACE2 binding. Prophylactic and therapeutic experiments in mice indicated that Nb4 could reduce the Omicron virus loads in the lung. In particular, in prophylactic experiments, intranasal administration of multivalent Nb4 completely protected mice from Omicron infection. Taken together, these results demonstrated that Nb4 could serve as a potent and broad-spectrum prophylactic and therapeutic Nb for COVID-19.

Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Proteins.

Green fluorescent proteins (GFPs) have lightened up almost every aspect of biological research including protein sciences. In the field of membrane protein structural biology, GFPs have been used widely to monitor membrane protein localization, expression level, the purification process and yield, and the stability inside the cells and in the test tube. Of particular interest is the fluorescence-detector size-exclusion chromatography-based thermostability assay (FSEC-TS). By simple heating and FSEC, the generally applicable method allows rapid assessment of the thermostability of GFP-fused membrane proteins without purification. Here we describe the experimental details and some typical results for the FSEC-TS method.

The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface.

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 (SaPgsA) 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.

Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody.

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-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD 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.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

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 high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2.

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 improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification.

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.

Thermostabilization of Membrane Proteins by Consensus Mutation: A Case Study for a Fungal Δ8-7 Sterol Isomerase.

Membrane proteins are generally challenging to work with because of their notorious instability. Protein engineering has been used increasingly to thermostabilize labile membrane proteins such as G-protein-coupled receptors for structural and functional studies in recent years. Two major strategies exist. Scanning mutagenesis systematically eliminates destabilizing residues, whereas the consensus approach assembles mutants with the most frequent residues among selected homologs, bridging sequence conservation with stability. Here, we applied the consensus concept to stabilize a fungal homolog of the human sterol Δ8-7 isomerase, a 26.4 kDa protein with five transmembrane helices. The isomerase is also called emopamil-binding protein (EBP), as it binds this anti-ischemic drug with high affinity. The wild-type had an apparent melting temperature (Tm) of 35.9 °C as measured by the fluorescence-detection size-exclusion chromatography-based thermostability assay. A total of 87 consensus mutations sourced from 22 homologs gained expression level and thermostability, increasing the apparent Tm to 69.9 °C at the cost of partial function loss. Assessing the stability and activity of several systematic chimeric constructs identified a construct with an apparent Tm of 79.8 °C and two regions for function rescue. Further back-mutations of the chimeric construct in the two target regions yielded the final construct with similar apparent activity to the wild-type and an elevated Tm of 88.8 °C, totaling an increase of 52.9 °C. The consensus approach is effective and efficient because it involves fewer constructs compared with scanning mutagenesis. Our results should encourage more use of the consensus strategy for membrane protein thermostabilization.

High-level heterologous expression of the human transmembrane sterol Δ8,Δ7-isomerase in Pichia pastoris.

Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P. pastoris, reaching ~200 mg L-1 in standard flasks, and ~1,000 mg L-1 in condensed culture that mimics fermentation. The heterogeneously expressed isomerase could be extracted fully with dodecyl maltoside, and the solubilized protein in the form of GFP fusion showed a sharp and symmetric peak on fluorescence-detection size exclusion chromatography. Our work provides a useful source for the purification of the recombinant isomerase.