COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our 2.85 Å cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. LA binding stabilizes a locked S conformation giving rise to reduced ACE2 interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies targeting LA binding by SARS-CoV-2.

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3 -terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3 -overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd ‫؍‬ 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd ‫؍‬ 3-5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.T elomeres are specialized DNA-protein complexes at the end of eukaryotic chromosomes. They protect the chromosome ends from recombination, from fusion, and from being mistaken as broken ends (1-3). Telomeric DNA consists of simple repetitive DNA sequences ranging in size from 36 nucleotides in hypotrichous ciliates up to 50 kb in mammals. The 3Ј-strand is usually G-rich and extends over the complementary strand. It has been shown that the G-rich overhang can adopt a variety of unusual DNA structures (4), of which guanine-quadruplex DNA and t-loops are stable in vitro under physiological conditions (5-8). Parallel-stranded as well as antiparallel-stranded guaninequadruplex structures have been biophysically and structurally analyzed in detail with synthetic oligonucleotides (6-11). It has been suggested that this structure is also involved in numerous cellular processes, including transcription and recombination, in addition to telomere function (12). However, direct evidence for this DNA structure in vivo has been lacking to date.Ciliated protozoa are a well-suited biological system to study telomere structure and function, and many important processes including telomere sequences and telomerase were first identified in these cells (1-3). Hypotrichous ciliates, such as Oxytricha, Euplotes, or Styl...

Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression. Here we describe new transfer vectors and a combination of DNA recombination-based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP-mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.

Secreted and membrane proteins are translocated across/into cell membranes via a proteinconducting channel (PCC). We present a cryo-EM reconstruction of the E. coli PCC, SecYEG, complexed with the ribosome and a signal anchor containing nascent chain, showing mRNA, three tRNAs, the nascent chain, and detailed features of both a translocating PCC and a second, nontranslocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEβ structure into the PCC EM densities favors a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. Based on our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.