Understanding the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites will inform development of improved therapeutics. We determined the structures of two human monoclonal antibodies AZD8895 and AZD1061, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2, in order to define the genetic and structural basis of neutralization. AZD8895 forms an “aromatic cage” at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1, and HCDR3 make interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germline-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.

We used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to characterize the conformational motion that couples energy expenditure to substrate translocation in the multidrug transporter MsbA. In liposomes, ligand-free MsbA samples conformations that depart from the crystal structures, including looser packing and water penetration along the periplasmic side. Adenosine triphosphate (ATP) binding closes the substrate chamber to the cytoplasm while increasing hydration at the periplasmic side, consistent with an alternating access model. Accentuated by ATP hydrolysis, the changes in the chamber dielectric environment and its geometry provide the likely driving force for flipping amphipathic substrates and a potential exit pathway. These results establish the structural dynamic basis of the power stroke in multidrug-resistant ATP-binding cassette (MDR ABC) transporters.

Astroviruses are single-stranded, plus-sense RNA viruses that infect both mammals and birds, causing gastroenteritis and other extraintestinal diseases. Clinical studies have established astroviruses as the second leading cause of viral diarrhea in young children. Here we report the crystal structure of the human astrovirus dimeric surface spike determined to 1.8-Å resolution. The overall structure of each spike/projection domain has a unique three-layered β-sandwiches fold, with a core, six-stranded β-barrel structure that is also found in the hepatitis E virus capsid protrusions, suggesting a closer phylogenetic relationship between these two viruses than previously acknowledged. Based on a hepatitis E virus capsid model, we performed homology modeling and produced a complete, T ¼ 3 astrovirus capsid model with features remarkably similar to those observed in a cryoelectron microscopy reconstruction image of a human astrovirus. Mapping conserved residues onto the astrovirus projection domain revealed a putative receptor binding site with amino acid compositions characteristic for polysaccharide recognition. Our results will have an important impact on future characterization of astrovirus structure and function, and will likely have practical applications in the development of vaccines and antivirals.structural protein | naked virus | attachment domain A stroviruses are small, nonenveloped, icosahedral viruses with a positive-sense, single-stranded RNA genome of approximately 7 kb in size. With members infecting mammals and avian species, the family of Astroviridae consists of two genera, Mammastrovirus and Avastrovirus (1, 2). Human astroviruses, eight serotypes in total, are one of the leading causes of gastroenteritis in young children, elderly people, and immunocompromised adults. Sporadic astrovirus infections as well as large-scale outbreaks in susceptible populations have been described (2). In addition to humans, astroviruses can infect a wide range of wild and domestic animals, causing gastroenteritis in most mammals and both intestinal and extraintestinal diseases in birds (1).The genomic RNA of astroviruses is polyadenylated with three ORFs. ORF1a and the downstream overlapping ORF1b encode two nonstructural polyproteins, nsp1a and nsp1ab. The nsp1ab protein is produced from both ORF1a and ORF1b through a translational frame-shift mechanism (3). Polyproteins nsp1a and nsp1ab are processed by a viral 3C-like serine protease and other cellular proteases into several proteins (e.g., the RNAdependent RNA polymerase and the serine protease) that are likely to function in the replication of the viral genome (4). ORF2 at the 3′ end of the viral genome encodes the viral capsid protein (CP) (5) and is found in both genomic and subgenomic RNAs. It is unclear whether the astrovirus RNA contains a 5′ cap. Viral capping enzymes such as guanylyltranferase and methyltransferases have not been identified in the astrovirus genome.When overexpressed in eukaryotic hosts, the 796-aa CP of the serotype 2 astrovirus was ...

Mammalian small heat shock proteins (sHSP) form polydisperse and dynamic oligomers that undergo equilibrium subunit exchange. Current models of their chaperone activity hypothesize that recognition and binding of protein non-native states involve changes in the oligomeric state. The equivalent thermodynamic representation is a set of three coupled equilibria that includes the sHSP oligomeric equilibrium, the substrate folding equilibrium, and the equilibrium binding between the sHSP and the substrate non-native states. To test this hypothesis and define the binding-competent oligomeric state of human Hsp27, we have perturbed the two former equilibria and quantitatively determined the consequences on binding. The substrate is a set of T4 lysozyme (T4L) mutants that bind under conditions that favor the folded state over the unfolded state by 10 2 -10 4 -fold. The concentration-dependent oligomer equilibrium of Hsp27 was perturbed by mutations that alter the relative stability of two major oligomeric states including phosphorylation-mimicking mutations that result in the dissociation to a small multimer over a wide range of concentrations. Correlation of binding isotherms with size exclusion chromatography analysis of the Hsp27 oligomer equilibrium demonstrates that the multimer is the binding-competent state. Binding occurs through two modes, each characterized by different affinity and number of binding sites, and results in T4L⅐Hsp27 complexes of different hydrodynamic properties. Mutants of the Hsp27 phosphorylation mimic that reverse the reduction in oligomer size also reduce the extent of T4L binding. Taken together, these results suggest a central role for the oligomeric equilibrium in regulating the chaperone activity of sHSP. The mutants identify sequence features important for modulating this equilibrium.

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an “aromatic cage” at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1–4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.