Tiamulin-Resistant Mutants of the Thermophilic Bacterium \u3cem\u3eThermus thermophilus\u3c/em\u3e

Tiamulin is a semisynthetic pleuromutilin antibiotic that binds to the 50S ribosomal subunit A site and whose (((2-diethylamino)ethyl)thio)-acetic acid tail extends into the P site to interfere with peptide bond formation. We have isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA. These mutations are consistent with those found in other organisms and are in close proximity to the crystallographically determined tiamulin binding site. We also conducted a cross-resistance analysis of nine other single-base substitutions in or near the peptidyltransferase active site, previously selected for resistance to structurally unrelated antibiotics. While some of the base substitutions in 23S rRNA are positioned to directly affect tiamulin-ribosome contacts, others are some distance from the tiamulin binding site, indicating an indirect mechanism of resistance. Similarly, amino acid substitutions in uL3 are predicted to act indirectly by destabilizing rRNA conformation in the active site. We interpret these observations in light of the available ribosome X-ray crystal structures. These results provide a more comprehensive profile of tiamulin resistance caused by mutations in the bacterial ribosome

Cryo-electron Microscopy Structure of the 70S Ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalisnow extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen

Cryo‑electron microscopy structure of the 70S ribosome from Enterococcus faecalis

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen

Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

Abstract The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell by inserting new copies, generating DNA damage, and triggering inflammation. Therefore, L1 inhibition could be used to treat many diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase due to the prevalence of well-characterized inhibitors of related viral enzymes. Here we present the L1 endonuclease as another target for reducing L1 activity. We characterize structurally diverse small molecule endonuclease inhibitors using computational, biochemical, and biophysical methods. We also show that these inhibitors reduce L1 retrotransposition, L1-induced DNA damage, and inflammation reinforced by L1 in senescent cells. These inhibitors could be used for further pharmacological development and as tools to better understand the life cycle of this element and its impact on disease processes

Tiamulin-Resistant Mutants of the Thermophilic Bacterium Thermus thermophilus

Tiamulin is a semisynthetic pleuromutilin antibiotic that binds to the 50S ribosomal subunit A site and whose (((2-diethylamino)ethyl)thio)-acetic acid tail extends into the P site to interfere with peptide bond formation. We have isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA. These mutations are consistent with those found in other organisms and are in close proximity to the crystallographically determined tiamulin binding site. We also conducted a cross-resistance analysis of nine other single-base substitutions in or near the peptidyltransferase active site, previously selected for resistance to structurally unrelated antibiotics. While some of the base substitutions in 23S rRNA are positioned to directly affect tiamulin-ribosome contacts, others are some distance from the tiamulin binding site, indicating an indirect mechanism of resistance. Similarly, amino acid substitutions in uL3 are predicted to act indirectly by destabilizing rRNA conformation in the active site. We interpret these observations in light of the available ribosome X-ray crystal structures. These results provide a more comprehensive profile of tiamulin resistance caused by mutations in the bacterial ribosome

The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (<b>1</b>) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (<b>2</b>), involving the transient generation and rearrangement of a neopentyl cation. In <i>Streptomyces arenae</i> this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologs being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate <b>2</b>, product <b>1</b>, and substrate analogue 6,7-dihydropentalenolactone F (<b>7</b>) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologs and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue <b>7</b> did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of <b>2</b>. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3_<i>re</i> of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-•Fe³⁺–OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1_<i>si</i>, which is flanked on each neighboring carbon by <i>syn</i>-axial substituents

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles

Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 Å resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners

Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9