Analysis of the 2.4-Å resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2 OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3 terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures. It is well known that single-stranded RNAs fold back on themselves to form short, double-stranded helices that are stabilized primarily by Watson-Crick and GU wobble base pairs. In recent years, as increasing numbers of RNA structures have been determined, additional, rarer elements of RNA secondary structure (1, 2) have been identified such as tetraloops (3, 4), bulged-G motifs (5-7), and cross-stand purine stacks (5,7,8).Less is known about the ways RNAs with complex secondary structures fold to form RNA tertiary structure because few of the RNA structures known previously were large enough to have sufficient tertiary structure to analyze that problem. In contrast, the recently determined structures of the large ribosomal subunit from Haloarcula marismortui (9, 10) and the small ribosomal subunit from Thermus thermophilus (11, 12) contain a large number of long-range interactions between regions of RNA that are distant in the secondary structure. The Ϸ3,000 nt of the two RNAs of the large ribosomal subunit form a compact structure stabilized by tertiary interactions between secondary structure elements that include about 100 double helical stems. The structure of this large polyanion is stabilized, in part, by interactions with metal ions and proteins, which will be discussed elsewhere. Here we address the interactions occurring between and among RNA helices and single strands that stabilize RNA tertiary and quaternary structure. MethodsFor our study, each adenosine residue in the structure of the H. marismortui 23S rRNA (Protein DataBank entry 1FFK) was assessed for occurrences of A-minor interactions by using the graphics program O (13). A-minor interactions were selected based on the following predetermined geometric criteria. The C2 atom of the adenosine had to be within 3.7 Å of one of its neighboring atoms. The interacting atom had to lie within 45°of the adenine plane. Finally, the C2 face of the adenosine had t...

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.

Starting from our previous finding of 14 known drugs as inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC50 values in a kinetic assay. Free-energy perturbation (FEP) calculations for Mpro-ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to Mpro. Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment of COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization.

The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

No abstract

A consensus virtual screening protocol has been applied to ca. 2000 approved drugs to seek inhibitors of the main protease (M pro ) of SARS-CoV-2, the virus responsible for COVID-19. 42 drugs emerged as top candidates, and after visual analyses of the predicted structures of their complexes with M pro , 17 were chosen for evaluation in a kinetic assay for M pro inhibition. Remarkably 14 of the compounds at 100-μM concentration were found to reduce the enzymatic activity and 5 provided IC 50 values below 40 μM: manidipine (4.8 μM), boceprevir (5.4 μM), lercanidipine (16.2 μM), bedaquiline (18.7 μM), and efonidipine (38.5 μM). Structural analyses reveal a common cloverleaf pattern for the binding of the active compounds to the P1, P1′, and P2 pockets of M pro . Further study of the most active compounds in the context of COVID-19 therapy is warranted, while all of the active compounds may provide a foundation for lead optimization to deliver valuable chemotherapeutics to combat the pandemic.

The crystal structure of an HIV-1 transactivation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-Å resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the inf luence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T in vivo.

The Klebsiellu aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174,4324-4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic &strand and exhibits unusually tight binding to phenyl-Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (M, = 35,000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 * 1.3 p M ) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(I1) in pseudo-octahedral geometry with 3-5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X-ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.