The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption 1,2 . There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

Abstract3D domain swapping is a mechanism for forming oligomeric proteins from their monomers. In 3D domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical protein chain. The result is an intertwined dimer or higher oligomer, with one domain of each subunit replaced by the identical domain from another subunit. The swapped "domain" can be as large as an entire tertiary globular domain, or as small as an a-helix or a strand of a P-sheet. Examples of 3D domain swapping are reviewed that suggest domain swapping can serve as a mechanism for functional interconversion between monomers and oligomers, and that domain swapping may serve as a mechanism for evolution of some oligomeric proteins. Domain-swapped proteins present examples of a single protein chain folding into two distinct structures.Keywords: aggregation; complementation; oligomer evolution; protein dimerization Since Svedberg's discovery of functional molecules composed of two or more identical protein chains, much effort has been expended in studying their metabolic regulation (Monod et al., 1965;Koshland et al., 1966) and their assembly and disassembly (Kikuchi & King, 1975;Caspar, 1980;Jaenicke, 1995).Despite this progress, understanding the assembly of oligomeric proteins from monomers remains a challenge. A common observation is that disassembly of an oligomeric protein into its monomeric subunits is accompanied by irreversible unfolding and aggregation. This observation is often interpreted in terms of exposing apolar patches on the monomer surface that are covered in the oligomer, thereby providing binding energy from a hydrophobic interaction. Thus, the question remains of how the oligomer could have been assembled in the first place. We propose an answer to this question for some oligomers based on a mode of association that we have noticed in several proteins of Reprint requests to: David Eisenberg, Molecular Biology Institute, Department of Chemistry and Biochemistry, and UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, University of California-Los Angeles, Los Angeles, California 90095-1570; e-mail: david@pauling.rnbi.ucla.edu.Abbreviations: BS-RNase, bovine seminal ribonuclease; DT, diphtheria toxin; GM-CSF, granulocyte-macrophage colony-stimulating factor; GST, glutathione S-transferase; IF, interferon; IL, interleukin; RNase, ribonuclease. known structure. We term this mode of association 3 0 domain swapping, because oligomers are formed from stable monomers by exchanging domains.A problem related to the formation of oligomeric proteins in a cell is the problem of how oligomeric proteins evolved from monomeric precursor proteins. For an oligomer to evolve, random mutations must change the surface of the monomer so that sufficient free energy is released upon oligomerization to overcome the accompanying entropy loss of immobilizing the monomers. As we discuss in this review, single amino acid replacements must be fortuitous to provide an adequate free energy of interaction. But an ...

The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.

ABSTRACTAn outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

The crystal structure of the diphtheria toxin dimer at 2.5 A resolution reveals a Y-shaped molecule of three domains. The catalytic domain, called fragment A, is of the alpha + beta type. Fragment B actually consists of two domains. The transmembrane domain consists of nine alpha-helices, two pairs of which are unusually apolar and may participate in pH-triggered membrane insertion and translocation. The receptor-binding domain is a flattened beta-barrel with a jelly-roll-like topology. Three distinct functions of the toxin, each carried out by a separate structural domain, can be useful in designing chimaeric proteins, such as immunotoxins, in which the receptor-binding domain is substituted with antibodies to target other cell types.

HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.

The comparison of monomeric and dimeric diphtheria toxin (DT) reveals a mode for protein aciation which we call domain swapping. The structure of dimeric DT has been extensively refined against data to 2.0-A resoutin and a three-residue loop has been co d as compared with our published 2.5-A-resolution s re. The monomeric DT structure has also been determined, at 2.3-A resolution. Monomeric DT is a Y-shaped molecule with three domains: catalytic (C), transmembrane (T), and receptor binding (R). Upon fwzing in phosphate buffer, DT forms a long-lived, metstable dimer. The protein chain tracing doses that upon dim tion an unprecedented conformational rerangement occurs: the entire R domain from each molecule of the dimer is excag for the R domain from the other. This involves breaking the noncovalent interactions between the R domain and the C and T domai, rotating the R domain by 180°with atomic movements up to 65 A, and re-forming the same noncovalent interactions between the R domain and the C and T do of the other chain of the dimer. This conformational in exa the long life and metastability of the DT dimer. Several other intrtwined, dimeric protein strucres sadisf our definition of domain swapping and suggest that domain swapping may be the molecular mechanism for evolution of these oligomers and possibly of oligomeric proteins in general.

HFE is related to major histocompatibility complex (MHC) class I proteins and is mutated in the iron-overload disease hereditary haemochromatosis. HFE binds to the transferrin receptor (TfR), a receptor by which cells acquire iron-loaded transferrin. The 2.8 A crystal structure of a complex between the extracellular portions of HFE and TfR shows two HFE molecules which grasp each side of a twofold symmetric TfR dimer. On a cell membrane containing both proteins, HFE would 'lie down' parallel to the membrane, such that the HFE helices that delineate the counterpart of the MHC peptide-binding groove make extensive contacts with helices in the TfR dimerization domain. The structures of TfR alone and complexed with HFE differ in their domain arrangement and dimer interfaces, providing a mechanism for communicating binding events between TfR chains. The HFE-TfR complex suggests a binding site for transferrin on TfR and sheds light upon the function of HFE in regulating iron homeostasis.