Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design.

Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.

Conformational changes of elongation factor G on the ribosome during tRNA translocation.

The universally conserved GTPase elongation factor G (EF-G) catalyzes the translocation of tRNA and mRNA on the ribosome after peptide bond formation. Despite numerous studies suggesting that EF-G undergoes extensive conformational rearrangements during translocation, high-resolution structures exist for essentially only one conformation of EF-G in complex with the ribosome. Here, we report four atomic-resolution crystal structures of EF-G bound to the ribosome programmed in the pre- and posttranslocational states and to the ribosome trapped by the antibiotic dityromycin. We observe a previously unseen conformation of EF-G in the pretranslocation complex, which is independently captured by dityromycin on the ribosome. Our structures provide insights into the conformational space that EF-G samples on the ribosome and reveal that tRNA translocation on the ribosome is facilitated by a structural transition of EF-G from a compact to an elongated conformation, which can be prevented by the antibiotic dityromycin.

Structures of the Cmr-ß Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.

LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice.

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.

Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization.

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

NOP14-mediated ribosome biogenesis is required for mTORC2 activation and predicts rapamycin sensitivity.

The mechanistic target of rapamycin (mTOR) forms two distinct complexes: rapamycin-sensitive mTOR complex 1 (mTORC1) and rapamycin-insensitive mTORC2. mTORC2 primarily regulates cell survival by phosphorylating Akt, though the upstream regulation of mTORC2 remains less well-defined than that of mTORC1. In this study, we show that NOP14, a 40S ribosome biogenesis factor and a target of the mTORC1-S6K axis, plays an essential role in mTORC2 signaling. Knockdown of NOP14 led to mTORC2 inactivation and Akt destabilization. Conversely, overexpression of NOP14 stimulated mTORC2-Akt activation and enhanced cell proliferation. Fractionation and coimmunoprecipitation assays demonstrated that the mTORC2 complex was recruited to the rough endoplasmic reticulum through association with endoplasmic reticulum-bound ribosomes. In vivo, high levels of NOP14 correlated with poor prognosis in multiple cancer types. Notably, cancer cells with NOP14 high expression exhibit increased sensitivity to mTOR inhibitors, because the feedback activation of the PI3K-PDK1-Akt axis by mTORC1 inhibition was compensated by mTORC2 inhibition partly through NOP14 downregulation. In conclusion, our findings reveal a spatial regulation of mTORC2-Akt signaling and identify ribosome biogenesis as a potential biomarker for assessing rapalog response in cancer therapy.

Structural basis of a two-step tRNA recognition mechanism for plastid glycyl-tRNA synthetase.

Two types of glycyl-tRNA synthetase (GlyRS) are known, the α2 and the α2ß2 GlyRSs. Both types of synthetase employ a class II catalytic domain to aminoacylate tRNAGly. In plastids and some bacteria, the α and ß subunits are fused and are designated as (αß)2 GlyRSs. While the tRNA recognition and aminoacylation mechanisms are well understood for α2 GlyRSs, little is known about the mechanisms for α2ß2/(αß)2 GlyRSs. Here we describe structures of the (αß)2 GlyRS from Oryza sativa chloroplast by itself and in complex with cognate tRNAGly. The set of structures reveals that the U-shaped ß half of the synthetase selects the tRNA in a two-step manner. In the first step, the synthetase engages the elbow and the anticodon base C35 of the tRNA. In the second step, the tRNA has rotated ∼9° toward the catalytic centre. The synthetase probes the tRNA for the presence of anticodon base C36 and discriminator base C73. This intricate mechanism enables the tRNA to access the active site of the synthetase from a direction opposite to that of most other class II synthetases.

Structures and implications of the C962R protein of African swine fever virus.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.

Annealing synchronizes the 70S ribosome into a minimum-energy conformation.

Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70S ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.

RplI interacts with 5' UTR of exsA to repress its translation and type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5' untranslated region (5' UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5' UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa.

Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.

A type III-A CRISPR-Cas system mediates co-transcriptional DNA cleavage at the transcriptional bubbles in close proximity to active effectors.

Many type III CRISPR-Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.

A scalable system for the fast production of RNA with homogeneous terminal ends.

In vitro transcription (IVT) using T7 RNA polymerase has become the most common method to synthesize RNAs for use in basic research and pharmaceutical applications. To solve the heterogeneity issue associated with the system, cis-acting ribozymes have been exploited to direct co-transcriptional processing to yield target RNAs with precisely defined ends. However, traditionally used ribozymes have many limitations, such as low efficiency and special sequence requirements of target RNAs. In addition, the introduction of ribozymes in IVT complicates the downstream purification of target RNAs. Here we describe a new cassette of engineered ribozymes (HHV-Kt and Twister-Kt) that can work in concert to produce RNA with defined 5' and 3' ends. The pair of ribozymes displayed reliably high activity when working with RNA of various lengths and structures. The engineered ribozymes also carry a K-turn RNA motif that enables fast post-IVT clearance of cleaved ribozymes and uncleaved precursors using K-turn affinity resins. Finally, we demonstrated the scalability of our system for the rapid production of large quantities of homogeneous RNA samples.

DNA targeting by subtype I-D CRISPR-Cas shows type I and type III features.

Prokaryotic CRISPR-Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR-Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3' and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR-Cas systems.

Molecular basis of the multifaceted functions of human leucyl-tRNA synthetase in protein synthesis and beyond.

Human cytosolic leucyl-tRNA synthetase (hcLRS) is an essential and multifunctional enzyme. Its canonical function is to catalyze the covalent ligation of leucine to tRNALeu, and it may also hydrolyze mischarged tRNAs through an editing mechanism. Together with eight other aminoacyl-tRNA synthetases (AaRSs) and three auxiliary proteins, it forms a large multi-synthetase complex (MSC). Beyond its role in translation, hcLRS has an important moonlight function as a leucine sensor in the rapamycin complex 1 (mTORC1) pathway. Since this pathway is active in cancer development, hcLRS is a potential target for anti-tumor drug development. Moreover, LRS from pathogenic microbes are proven drug targets for developing antibiotics, which however should not inhibit hcLRS. Here we present the crystal structure of hcLRS at a 2.5 Å resolution, the first complete structure of a eukaryotic LRS, and analyze the binding of various compounds that target different sites of hcLRS. We also deduce the assembly mechanism of hcLRS into the MSC through reconstitution of the entire mega complex in vitro. Overall, our study provides the molecular basis for understanding both the multifaceted functions of hcLRS and for drug development targeting these functions.

Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus.

Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2αHD and Cmr2αPalm mutants, but the inhibition was diminished in Cmr2αHD,Palm. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.

High-resolution DNA quadruplex structure containing all the A-, G-, C-, T-tetrads.

DNA can form diverse structures, which predefine their physiological functions. Besides duplexes that carry the genetic information, quadruplexes are the most well-studied DNA structures. In addition to their important roles in recombination, replication, transcription and translation, DNA quadruplexes have also been applied as diagnostic aptamers and antidisease therapeutics. Herein we further expand the sequence and structure complexity of DNA quadruplex by presenting a high-resolution crystal structure of DNA1 (5'-AGAGAGATGGGTGCGTT-3'). This is the first quadruplex structure that contains all the internal A-, G-, C-, T-tetrads, A:T:A:T tetrads and bulged nucleotides in one single structure; as revealed by site-specific mutagenesis and biophysical studies, the central ATGGG motif plays important role in the quadruplex formation. Interestingly, our structure also provides great new insights into cation recognition, including the first-time reported Pb2+, by tetrad structures.

Cmr1 enables efficient RNA and DNA interference of a III-B CRISPR-Cas system by binding to target RNA and crRNA.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems provide adaptive immunity against invasive nucleic acids guided by CRISPR RNAs (crRNAs) in archaea and bacteria. Type III CRISPR-Cas effector complexes show RNA cleavage and RNA-activated DNA cleavage activity, representing the only known system of dual nucleic acid interference. Here, we investigated the function of Cmr1 by genetic assays of DNA and RNA interference activity in the mutants and biochemical characterization of their mutated Cmr complexes. Three cmr1α mutants were constructed including ΔßΔ1α, Δß1α-M1 and Δß1α-M2 among which the last two mutants carried a double and a quadruple mutation in the first α-helix region of Cmr1α. Whereas the double mutation of Cmr1α (W58A and F59A) greatly influenced target RNA capture, the quadruple mutation almost abolished crRNA binding to Cmr1α. We found that Cmr2α-6α formed a stable core complex that is active in both RNA and DNA cleavage and that Cmr1α strongly enhances the basal activity of the core complex upon incorporation into the ribonucleoprotein complex. Therefore, Cmr1 functions as an integral activation module in III-B systems, and the unique occurrence of Cmr1 in III-B systems may reflect the adaptive evolution of type III CRISPR-Cas systems in thermophiles.

Box C/D guide RNAs recognize a maximum of 10 nt of substrates.

Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D' and boxes C' to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide-substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C' side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.

Elongation factor 4 remodels the A-site tRNA on the ribosome.

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5'-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.