CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding.

Simple Secondary Amines Inhibit Growth of Gram-Negative Bacteria through Highly Selective Binding to Phenylalanyl-tRNA Synthetase.

Antibiotics to treat drug-resistant Gram-negative infections are urgently needed but challenging to discover. Using a cell-based screen, we identified a simple secondary amine that inhibited the growth of wild-type and but not the growth of the Gram-positive organism . Resistance mutations in and mapped exclusively to the aminoacyl-tRNA synthetase PheRS.

Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking.

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain.

Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization.

Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix.

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses.

Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport.

ATP-binding cassette (ABC) transporters constitute a large family of proteins present in all domains of life. They are powered by dynamic ATPases that harness energy from binding and hydrolyzing ATP through a cycle that involves the closing and reopening of their two ATP-binding domains. The LptBFGC exporter is an essential ABC transporter that assembles lipopolysaccharides (LPS) on the surface of Gram-negative bacteria to form a permeability barrier against many antibiotics.

Structural basis of unidirectional export of lipopolysaccharide to the cell surface.

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide. This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface.

Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport.

Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness.

Substrate Preferences Establish the Order of Cell Wall Assembly in Staphylococcus aureus.

The Gram-positive bacterial cell wall is a large supramolecular structure and its assembly requires coordination of complex biosynthetic pathways. In the step that merges the two major biosynthetic pathways in Staphylococcus aureus cell wall assembly, conserved protein ligases attach wall teichoic acids to peptidoglycan, but the order of biosynthetic events is a longstanding question. Here, we use a chemical approach to define which of the possible peptidoglycan intermediates are substrates for wall-teichoic acid ligases, thereby establishing the order of cell wall assembly.

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin.

Antibiotic That Inhibits the ATPase Activity of an ATP-Binding Cassette Transporter by Binding to a Remote Extracellular Site.

Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230 000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

Unlabelled: The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface.