Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

The HIV-1 p66 homodimeric RT exhibits different conformations in the binding-competent and -incompetent NNRTI site.

Non-nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase (RT), NNRTIs, which bind to the p66/p51 heterodimeric RT, also interact with the p66/p66 homodimer, whose structure is unknown. 19 F nuclear magnetic resonance of a single 4-trifluoromethylphenylalanine (tfmF) residue, incorporated into the NNRTI binding pocket of the p66/p66 homodimer at position 181, was used to investigate NNRTI binding. In the NNRTI-bound homodimer complex, two different 19 F signals are observed, with the resonance frequencies matching those of the NNRTI-bound p66/p51 heterodimer spectra, in which the individual p66-subunit or p51-subunit were labeled with tfmF at positions 181. These data suggest that the NNRTI-bound p66/p66 homodimer conformation, particularly around residue 181, is very similar to that in the p66/p51 heterodimer, explaining why NNRTI binding to p66/p66 enhances dimer formation.

Correction to: NMR structure of the HIV-1 reverse transcriptase thumb subdomain.

In the original publication of the article, the given name and family name of the author P. Andrew Karplus was published incorrectly. The name should read as "P. Andrew" - Given name and "Karplus" - Family name.

Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study.

HIV-1 reverse transcriptase (RT) is a major drug target in the treatment of HIV-1 infection. RT inhibitors currently in use include non-nucleoside, allosteric RT inhibitors (NNRTIs), which bind to a hydrophobic pocket, distinct from the enzyme's active site. We investigated RT-NNRTI interactions by solution (19)F nuclear magnetic resonance (NMR), using singly (19)F-labeled RT proteins. Comparison of (19)F chemical shifts of fluorinated RT and drug-resistant variants revealed that the fluorine resonance is a sensitive probe for identifying mutation-induced changes in the enzyme. Our data show that in the unliganded enzyme, the NNRTI-binding pocket is highly plastic and not locked into a single conformation. Upon inhibitor binding, the binding pocket becomes rigidified. In the inhibitor-bound state, the (19)F signal of RT is similar to that of drug-resistant mutant enzymes, distinct from what is observed for the free state. Our results demonstrate the power of (19)F NMR spectroscopy to characterize conformational properties using selectively (19)F-labeled protein.

NMR structure of the HIV-1 reverse transcriptase thumb subdomain.

The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT) has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme reveals that the overall structures are very similar, with only two regions exhibiting local conformational differences. The C-terminal capping pattern of the αH helix is subtly different, and the loop connecting the αI and αJ helices in the p51 chain of the full-length p51/p66 heterodimeric RT differs from our NMR structure due to unique packing interactions in mature RT. Overall, our data show that the thumb subdomain folds independently and essentially the same in isolation as in its natural structural context.

The p66 immature precursor of HIV-1 reverse transcriptase.

In contrast to the wealth of structural data available for the mature p66/p51 heterodimeric human immunodeficiency virus type 1 reverse transcriptase (RT), the structure of the homodimeric p66 precursor remains unknown. In all X-ray structures of mature RT, free or complexed, the processing site in the p66 subunit, for generating the p51 subunit, is sequestered into a ß-strand within the folded ribonuclease H (RNH) domain and is not readily accessible to proteolysis, rendering it difficult to propose a simple and straightforward mechanism of the maturation step. Here, we investigated, by solution NMR, the conformation of the RT p66 homodimer. Our data demonstrate that the RNH and Thumb domains in the p66 homodimer are folded and possess conformations very similar to those in mature RT. This finding suggests that maturation models which invoke a complete or predominantly unfolded RNH domain are unlikely. The present study lays the foundation for further in-depth mechanistic investigations at the atomic level.

Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction.

NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

Volume exclusion and soft interaction effects on protein stability under crowded conditions.

Most proteins function in nature under crowded conditions, and crowding can change protein properties. Quantification of crowding effects, however, is difficult because solutions containing hundreds of grams of macromolecules per liter often interfere with the observation of the protein being studied. Models for macromolecular crowding tend to focus on the steric effects of crowders, neglecting potential chemical interactions between the crowder and the test protein. Here, we report the first systematic, quantitative, residue-level study of crowding effects on the equilibrium stability of a globular protein. We used a system comprising poly(vinylpyrrolidone)s (PVPs) of varying molecular weights as crowding agents and chymotrypsin inhibitor 2 (CI2) as a small globular test protein. Stability was quantified with NMR-detected amide (1)H exchange. We analyzed the data in terms of hard particle exclusion, confinement, and soft interactions. For all crowded conditions, nearly every observed residue experiences a stabilizing effect. The exceptions are residues for which stabilities are unchanged. At a PVP concentration of 100 g/L, the data are consistent with theories of hard particle exclusion. At higher concentrations, the data are more consistent with confinement. The data show that the crowder also stabilizes the test protein by weakly binding its native state. We conclude that the role of native-state binding and other soft interactions needs to be seriously considered when applying both theory and experiment to studies of macromolecular crowding.

Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies.

The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1-5 show therapeutic promise and are being evaluated clincally6-8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to "up" RBDs, (2) ACE2-blocking hNAbs that bind both "up" and "down" RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize "up" and "down" RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only "up" RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent "down" RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies.

Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1A and RBD epitopes. A 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on "up" RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

(19)F-modified proteins and (19)F-containing ligands as tools in solution NMR studies of protein interactions.

(19)F solution NMR is a powerful and versatile tool to study protein structure and protein-ligand interactions due to the favorable NMR characteristics of the (19)F atom, its absence in naturally occurring biomolecules, and small size. Protocols to introduce (19)F atoms into both proteins and their ligands are readily available and offer the ability to conduct protein-observe (using (19)F-labeled proteins) or ligand-observe (using (19)F-containing ligands) NMR experiments. This chapter provides two protocols for the (19)F-labeling of proteins, using an Escherichia coli expression system: (i) amino acid type-specific incorporation of (19)F-modified amino acids and (ii) site-specific incorporation of (19)F-modified amino acids using recombinantly expressed orthogonal amber tRNA/tRNA synthetase pairs. In addition, we discuss several applications, involving (19)F-modified proteins and (19)F-containing ligands.

A bioreactor for in-cell protein NMR.

The inside of the cell is a complex environment that is difficult to simulate when studying proteins and other molecules in vitro. We have developed a device and system that provides a controlled environment for nuclear magnetic resonance (NMR) experiments involving living cells. Our device comprises two main parts, an NMR detection region and a circulation system. The flow of medium from the bottom of the device pushes alginate encapsulated cells into the circulation chamber. In the chamber, the exchange of oxygen and nutrients occurs between the media and the encapsulated cells. When the media flow is stopped, the encapsulated cells fall back into the NMR detection region, and spectra can be acquired. We have utilized the bioreactor to study the expression of the natively disordered protein alpha-synuclein, inside Escherichia coli cells.