C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought.

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.

An Update on Crop ABA Receptors.

The hormone abscisic acid (ABA) orchestrates the plant stress response and regulates sophisticated metabolic and physiological mechanisms essential for survival in a changing environment. Plant ABA receptors were described more than 10 years ago, and a considerable amount of information is available for the model plant Arabidopsis thaliana. Unfortunately, this knowledge is still very limited in crops that hold the key to feeding a growing population. In this review, we summarize genomic, genetic and structural data obtained in crop ABA receptors. We also provide an update on ABA perception in major food crops, highlighting specific and common features of crop ABA receptors.

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA.

The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.

Drug Discovery for Thirsty Crops.

Following virtual screening and structure-based ligand optimization, researchers have developed opabactin (OP), an abscisic acid (ABA)-receptor agonist with tenfold greater in vivo activity than ABA. This new ligand surpasses previous agonists for its potency and bioactivity on staple crops. OP leads a new class of agrochemicals designed to protect crops from drought.

An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function.

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA-protein and protein-protein interactions.

Modification of the wobble uridine in bacterial and mitochondrial tRNAs reading NNA/NNG triplets of 2-codon boxes.

Posttranscriptional modification of the uridine located at the wobble position (U34) of tRNAs is crucial for optimization of translation. Defects in the U34 modification of mitochondrial-tRNAs are associated with a group of rare diseases collectively characterized by the impairment of the oxidative phosphorylation system. Retrograde signaling pathways from mitochondria to nucleus are involved in the pathophysiology of these diseases. These pathways may be triggered by not only the disturbance of the mitochondrial (mt) translation caused by hypomodification of tRNAs, but also as a result of nonconventional roles of mt-tRNAs and mt-tRNA-modifying enzymes. The evolutionary conservation of these enzymes supports their importance for cell and organismal functions. Interestingly, bacterial and eukaryotic cells respond to stress by altering the expression or activity of these tRNA-modifying enzymes, which leads to changes in the modification status of tRNAs. This review summarizes recent findings about these enzymes and sets them within the previous data context.

Enzymology of tRNA modification in the bacterial MnmEG pathway.

Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway.

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.